Solid Substrates for Promoting Cell and Tissue Growth

ABSTRACT

This invention provides solid substrates for promoting cell or tissue growth or restored function, which solid substrate is characterized by a specific fluid uptake capacity value of at least 75%, which specific fluid uptake capacity value is determined by establishing a spontaneous fluid uptake value divided by a total fluid uptake value. This invention also provides solid substrates for promoting cell or tissue growth or restored function, which solid substrate is characterized by having a contact angle value of less than 60 degrees, when in contact with a fluid. This invention also provides solid substrates for promoting cell or tissue growth or restored function, which said substrate is characterized by a substantial surface roughness (Ra) as measured by scanning electron microscopy or atomic force microscopy. The invention also provides for processes for selection of an optimized coral-based solid substrate for promoting cell or tissue growth or restored function and applications of the same.

CROSS REFERENCE TO RELATED APPLICATIONS

U.S. application Ser. No. 14/767,428, U.S. Provisional Application No.61/763,981, U.S. Provisional Application No. 61/763,985, U.S.Provisional Application No. 61/764,467, and U.S. Provisional ApplicationNo. 61/764,496, all of which were filed Feb. 13, 2013, as well as U.S.Provisional Application No. 61/773,219 and U.S. Provisional ApplicationNo. 61/773,228, both of which were filed Mar. 6, 2013 are all herebyincorporated by reference herein in their entirety.

BACKGROUND OF THE INVENTION

Tissue growth, regeneration and repair are often necessary to restorefunction and reconstruct the morphology of the tissue, for example, as aresult of exposure to trauma, neoplasia, abnormal tissue growth, aging,and others.

Synthetic materials have also used as a substrate for promoting ex-vivotissue assembly and repair, and similarly for restoring andreconstructing different tissues, for example for bone, for many years,with mixed success. Another possibility is autologous tissue grafting,although the supply of autologous tissue is limited and its collectionmay be painful, with the risk of infection, hemorrhage, cosmeticdisability, nerve damage, and loss of function. In addition, significantmorbidity is associated with autograft harvest sites. These problems maybe overcome by engineering tissue using solid substrates made ofsynthetic or natural biomaterials that promote the adhesion, migration,proliferation, and differentiation of stem cells, for example,mesenchymal stem cells (MSCs).

Many diseases and conditions whose treatment is sought would benefitfrom the ability to promote cell and tissue growth in a site-specificmanner, promoting growth and incorporation of new tissue within adamaged or diseased site.

In bone and cartilage applications, the immediate microenvironment andthe three-dimensional (3D) organization are important factors indifferentiation in general and particularly in chondrogenic andosteogenic differentiation.

Some bone tissue engineering scaffolds consists of natural polymers,such as collagen, alginate, hyaluronic acid, and chitosan. Naturalmaterials offer the advantages of specific cell interaction, easyseeding of cells because of their hydrophilic interactions, low toxicityand low chronic inflammatory response. However, these scaffolds oftenare mechanically unstable and do not readily contribute to the creationof tissue structures with a specific predefined shape fortransplantation. To obtain mechanical strength, chemical modification isrequired, which may lead to toxicity.

Defects and degeneration of the articular cartilage surfaces of jointscauses pain and stiffness. Damage to cartilage which protects joints canresult from either physical injury as a result of trauma, sports orrepetitive stresses (e.g., osteochondral fracture, secondary damage dueto cruciate ligament injury) or from disease (e.g. osteoarthritis,rheumatoid arthritis, aseptic necrosis, osteochondritis dissecans).

Osteoarthritis (OA) results from general wear and tear of joints, mostnotably hip and knee joints. Osteoarthritis is common in the elderlybut, in fact, by age 40 most individuals have some osteoarthitic changesin their weight bearing joints. Another emerging trend increasing theprevalence of osteoarthritis is the rise in obesity. The CDC estimatesthat 30% of American adults (or 60 million people) are obese. Obeseadults are 4 times more likely to develop knee OA than normal weightadults Rheumatoid arthritis is an inflammatory condition which resultsin the destruction of cartilage. It is thought to be, at least in part,an autoimmune disease with sufferers having a genetic predisposition tothe disease.

Orthopedic prevention and repair of damaged joints is a significantburden on the medical profession both in terms of expense and time spenttreating patients. In part, this is because cartilage does not possesthe capacity for self-repair. Attempts to re-grow hyaline cartilage forrepair of cartilage defects remain unsuccessful. Orthopedic surgery isavailable in order to repair defects and prevent articular damage in aneffort to forestall serious degenerative changes in a joint. The use ofsurgical techniques often requires the removal and donation of healthytissue to replace the damaged or diseased tissue. Techniques utilizingdonated tissue from autografts, allografts, or xenografts are whollyunsatisfactory as autografts add additional trauma to a subject andallografts and xenografts are limited by immunological reactivity to thehost subject and possible transfer of infective agents. Surgicalattempts to utilize materials other than human or animal tissue forcartilage regeneration have been unsuccessful.

More broadly, there is also a lack of appropriate solid substrates forother applications in cell and tissue growth, expansion and modeling, aswell.

An ideal material which restores tissue function and facilitatesreconstruction of the morphology of such tissue is as yet, lacking.

SUMMARY OF THE INVENTION

In some embodiments, the present invention provides optimized solidsubstrates for promoting cell or tissue growth or restored function. Insome embodiments, the invention provides a process for the selection ofan optimized marine organism skeletal derivative-based solid substratefor promoting cell or tissue growth or restored function, comprisingestablishing a specific fluid uptake capacity value for the marineorganism skeletal derivative-based solid material, and selecting amarine organism skeletal derivative-based solid material characterizedby a specific fluid uptake capacity value of at least 75%.

In some embodiments, the invention provides a solid substrate forpromoting cell or tissue growth or restored function, which solidsubstrate comprises a optimized marine organism skeletal derivative andis characterized by a specific fluid uptake capacity value of at least75%, which specific fluid uptake capacity value is determined byestablishing a spontaneous fluid uptake value divided by a total fluiduptake value and which solid substrate has been exposed to certainpost-isolation purification and/or processing procedures.

In some embodiments, the solid substrate in accordance with thisinvention and obtained via the processes of this invention ischaracterized by a specific fluid uptake capacity value of at least 80%,and in some embodiments, the solid substrate in accordance with thisinvention and obtained via the processes of this invention ischaracterized by a specific fluid uptake capacity value of at least 85%,and in some embodiments, the solid substrate in accordance with thisinvention and obtained via the processes of this invention ischaracterized by a specific fluid uptake capacity value of at least 90%,and in some embodiments, the solid substrate in accordance with thisinvention and obtained via the processes of this invention ischaracterized by a specific fluid uptake capacity value of at least 95%,and in some embodiments, the solid substrate in accordance with thisinvention and obtained via the processes of this invention ischaracterized by a specific fluid uptake capacity value of at least 97%.In some embodiments, the solid substrate in accordance with thisinvention and obtained via the processes of this invention ischaracterized by a specific fluid uptake capacity value of from 75-100%.

In some embodiments, the term “a specific fluid uptake capacity value”is also referred to herein as “SFUC” or “SWC”, all of which are to beunderstood to be interchangeable.

In some embodiments, a specific fluid uptake capacity value of thisinvention is determined using an apparatus, which is automated. In someaspects, and as exemplified and further described hereinunder,assessment of the specific fluid uptake capacity value of varioussamples may be simultaneously or sequentially assessed, as part of anautomated scaled-up process appropriate for commercial production. Insome aspects, such apparatus may further provide for the individualselection and transport of samples having desired characteristics.

In some embodiments, the invention provides a solid substrate forpromoting cell or tissue growth or restored function, or a process forobtaining same, which solid substrate comprises a organism skeletalderivative and is characterized by having a contact angle value of lessthan 60 degrees, when in contact with a fluid.

As the skilled artisan will appreciate, a contact angle may bedetermined as described and exemplified herein, using standardmethodology and equipment, for example, via goniometry. In someembodiments, such methods may make use of processes as described in P.A. Thomson, W. B. Brinckerhoff, M. O. Robbins, in: K. L. Mittal (Ed.),Contact Angle Wettability and Adhesion, VSP, Utrecht, 1993, pp. 139-158;E. L. Decker, S. Garof, Langmuir 13 (1997) 6321; and M. G. Orkoula etal.: Colloids and Surfaces A: Physicochem. Eng. Aspects 157 (1999)333-340; Hiemenz, P. C.; Rajagopalan, R. Principles of Colloid andSurface Chemistry, 1997, 3rd Ed., Marcel Dekker, Inc; Applied Colloidand Surface Chemistry Chapter 2: Surface Tension and wetting, by RichardPashley, Marilyn Karaman, 2004, John Wiley and sons, all of which arehereby incorporated in their entirety.

In some embodiments, the invention provides a solid substrate forpromoting cell or tissue growth or restored function, or a process forobtaining same, which solid substrate is characterized by substantialsurface roughness (Ra) as measured by scanning electron microscopy oratomic force microscopy.

In some embodiments, the coral or coral derivative is aragonite,calcite, mixtures thereof, or other polymorphs of the same.

In some embodiments the structure composition of the coral or coralderivative is determined by X-ray diffraction (XRD) or Feigl solutionpositive staining.

In some embodiments, the solid substrate is isolated from a Poritesspecies, a Goniopora, a Millepora species or an Acropora species.

In some embodiments, the solid substrate is isolated from a barnacle ormollusk. In some embodiments, the solid substrate is comprised of nacre.

In some embodiments, the invention provides a kit comprising one or moresolid substrates as herein described. In some embodiments, the kit willcomprise a series of solid substrates characterized by a specific fluiduptake capacity value of at least 75% and/or produced by a process ofthis invention, where the marine organism skeletal derivative-basedsolid materials in the kit have a specific fluid uptake capacity valueof from 75% to 99%, and in some embodiments, the kit contains a seriesof solid substrates characterized by a specific fluid uptake capacityvalue of from 80% to 99%, and in some embodiments, the kit contains aseries of solid substrates characterized by a specific fluid uptakecapacity value of from 85% to 99%, and in some embodiments, the kitcontains a series of solid substrates characterized by a specific fluiduptake capacity value of from 90% to 99%, and in some embodiments, thekit contains a series of solid substrates characterized by a specificfluid uptake capacity value of from 95% to 99% or, in other embodiments,from 95% to 100%.

In another embodiment, the invention provides a process for selection ofan optimized marine organism skeletal derivative-based solid substratefor promoting cell or tissue growth or restored function, said processcomprising:

-   -   isolating a marine organism skeletal derivative-based solid        material;    -   establishing a specific fluid uptake capacity value of said        marine organism skeletal derivative-based solid material, which        specific fluid uptake capacity value is determined by        establishing a spontaneous fluid uptake value divided by a total        fluid uptake value; and    -   selecting a marine organism skeletal derivative-based solid        material characterized by a specific fluid uptake capacity value        of at least 75%.

In some embodiments, according to this aspect, the process furthercomprises the step of contacting said marine organism skeletalderivative-based solid material with a fluid for from 0.1-15 minutes topromote spontaneous fluid uptake of said fluid within said marineorganism skeletal derivative-based solid material to arrive at saidspontaneous fluid uptake value.

In some embodiments, according to this aspect, the process furthercomprises the step of contacting said marine organism skeletalderivative-based solid material with a fluid and applying negativepressure to said marine organism skeletal derivative-based solidmaterial to promote maximal uptake of said fluid within said marineorganism skeletal derivative-based solid material to arrive at saidtotal fluid uptake value.

In some embodiments, according to this aspect, the specific fluid uptakecapacity value is a function of change in weight in said marine organismskeletal derivative-based solid material.

In some embodiments, according to this aspect, the change in weight insaid marine organism skeletal derivative-based solid material is due toabsorbance of said fluid within interstices in said solid material, orin some embodiments, due to absorbance of said fluid within pores insaid solid material, or in some embodiments, the change in weight insaid marine organism skeletal derivative-based solid material is due toabsorbance of said fluid within interstices in said solid material anddue to absorbance of said fluid within pores in said solid material,which in some embodiments, is within or in some embodiments, between,individual coral crystals.

In some embodiments, according to this aspect, the specific fluid uptakecapacity value is a function of change in fluid volume of applied fluidto said coralline-based solid material.

In some embodiments, this invention provides a solid substrate forpromoting cell or tissue growth or restored function, which solidsubstrate comprises an organism skeletal derivative and is characterizedby having a contact angle value of less than 60 degrees, when in contactwith a fluid.

In some embodiments, this invention provides a process for selection ofan optimized organism skeletal derivative-based solid substrate forpromoting cell or tissue growth or restored function, said processcomprising:

-   -   Isolating or preparing a organism skeletal derivative-based        solid material;    -   contacting said organism skeletal derivative-based solid        material with a fluid and establishing a contact angle for said        organism skeletal derivative; and    -   selecting a organism skeletal derivative-based solid material        characterized by a contact angle of less than 60 degrees.

In some embodiments, the processes of this invention, which facilitateselection of an optimized organism skeletal derivative-based solidsubstrate for promoting cell or tissue growth or restored function mayinclude a step whereby the contact of the organism skeletalderivative-based solid material with a fluid and establishing a contactangle for said organism skeletal derivative, or the establishing aspecific fluid uptake capacity value of said marine organism skeletalderivative-based solid material, which specific fluid uptake capacityvalue is determined by establishing a spontaneous fluid uptake valuedivided by a total fluid uptake value may be performed on samplesimmediately proximal to a sample of interest, and in some embodiments,from within a comparable region, for example, in terms of a region ofcoral growth in a growth ring, with the selection envisioned to be basedin some embodiments, on the performance of proximal regions, and theirachievement of the desired criteria for selection, as herein described.

In some embodiments, the marine organism skeletal derivative-based solidmaterial is substantially comprised of calcium carbonate.

In some embodiments the process further comprises the steps of:

-   -   establishing the presence of a substantially rough surface on        said marine organism skeletal derivative-based solid material,        which substantially rough surface is determined by scanning        electron microscopy or atomic force microscopy; and    -   selecting a marine organism skeletal derivative-based solid        material characterized by a determination of the presence of a        substantially rough surface on said marine organism skeletal        derivative-based solid material.

In some embodiments, the invention provides a process for selection ofan optimized organism skeletal derivative-based solid substrate forpromoting cell or tissue growth or restored function, said processcomprising:

-   -   establishing the presence of a substantially rough surface on        said marine organism skeletal derivative-based solid material,        which substantially rough surface is determined by scanning        electron microscopy or atomic force microscopy; and    -   selecting a marine organism skeletal derivative-based solid        material characterized by a determination of the presence of a        substantially rough surface on said marine organism skeletal        derivative-based solid material.

In some embodiments, the invention provides process for selection of anoptimized organism skeletal derivative-based solid substrate forpromoting cell or tissue growth or restored function, comprising acombination of the steps described for such processes as describedherein.

In some embodiments, a process of this invention further comprises thestep of contacting said solid substrate with cells or tissue.

In some embodiments, according to this aspect, the contacting promotesadhesion, proliferation or differentiation, or a combination thereof, ofsaid cells or cells within said tissue.

In some embodiments, a fluid is a protein-containing, salt-containing orcarbohydrate containing solution, or in some embodiments, the fluid is abiologic fluid, and in some embodiments, the biologic fluid isautologous or allogeneic with respect to a cell or tissue of a subjectwhen said solid substrate is contacted with a cell or tissue of saidsubject. In some embodiments, the fluid is water.

In some embodiments, the solid substrate promotes cell or tissue growthin tissue damaged by trauma or disease.

In some embodiments, the invention provides a solid substrate producedby the process according to any aspect as herein described.

In some embodiments, this invention provides a process for converting asuboptimal marine organism skeletal derivative-based solid substrate toan optimized marine organism skeletal derivative-based solid substratesfor promoting cell or tissue growth or restored function, said processcomprising:

-   -   a) establishing a specific fluid uptake capacity value for a        group of marine organism skeletal derivative-based solid        materials, which specific fluid uptake capacity value is        determined by establishing a spontaneous fluid uptake value        divided by a total fluid uptake value for each sample in said        group;    -   b) selecting a marine organism skeletal derivative-based solid        material characterized by a specific fluid uptake capacity        value;    -   c) contacting said marine organism skeletal derivative-based        solid material of (b) with an amphiphilic material, a polar        solvent, a cationic material, an anionic material, or a        combination thereof;    -   d) determining a specific fluid uptake capacity as in (a) in        said marine organism skeletal derivative-based solid materials        obtained in (c); and    -   e) selecting marine organism skeletal derivative-based solid        materials obtained in (d) having a newly established increased        specific fluid uptake capacity value.

This invention also provides a process for converting a suboptimalmarine organism skeletal derivative-based solid substrate to anoptimized marine organism skeletal derivative-based solid substrates forpromoting cell or tissue growth or restored function, said processcomprising:

-   -   a) establishing a specific fluid uptake capacity value for a        group of marine organism skeletal derivative-based solid        materials, which specific fluid uptake capacity value is        determined by establishing a spontaneous fluid uptake value        divided by a total fluid uptake value for each sample in said        group;    -   b) selecting a marine organism skeletal derivative-based solid        material characterized by a specific fluid uptake capacity        value;    -   c) subjecting said marine organism skeletal derivative-based        solid material of (b) to cold plasma treatment, corona        treatment, or a combination thereof;    -   d) determining a specific fluid uptake capacity as in (a) in        said marine organism skeletal derivative-based solid materials        obtained in (c); and    -   e) selecting marine organism skeletal derivative-based solid        materials obtained in (d) having a newly established increased        specific fluid uptake capacity value.

In some embodiments, the specific fluid uptake capacity value isincreased by at least 5%. In some embodiments, the selected marineorganism skeletal derivative-based solid material is characterized by aspecific fluid uptake capacity value of between 75% and 95%. In someembodiments, the increased specific fluid uptake capacity value isincreased by at least 15%. In some embodiments, the selected marineorganism skeletal derivative-based solid material is characterized by aspecific fluid uptake capacity value of between 45% and 70%. In someembodiments, the increased specific fluid uptake capacity value isincreased by at least 35%. In some embodiments, the selected marineorganism skeletal derivative-based solid material is characterized by aspecific fluid uptake capacity value of between 1% and 40%. In someembodiments, the solid substrate is comprised substantially of a coralor coral-based derivative. In some embodiments, the solid substrate iscomprised substantially of aragonite, calcite, hydroxyapatite or acombination thereof. In some embodiments, the process further comprisesthe step of fully or partially converting or coating a marine organismskeletal derivative-based solid material to hydroxyapatite prior toestablishing said specific fluid uptake capacity value in (d), whereinsaid marine organism skeletal derivative-based solid material isaragonite. In some embodiments, the process further comprises the stepof fully or partially converting a marine organism skeletalderivative-based solid material to hydroxyapatite subsequent toestablishing said specific fluid uptake capacity value in (d), whereinsaid marine organism skeletal derivative-based solid material is mostlycalcium carbonate. In some embodiments, the marine organism skeletalderivative-based solid material can be used as a bone filler or bonesubstitute material. In some embodiments, the amphiphilic material, apolar solvent, a cationic material or an anionic material is tween,pluronic, ethanol, methylene blue, hyaluronic acid, chondroitin sulfateor a combination thereof.

In some embodiments, the process further comprises the step of applyinga secondary cleansing method to said marine organism skeletalderivative-based solid materials of (b), following contact with saidamphiphilic material, polar solvent, cationic material, anionicmaterial, or combinations thereof. In some embodiments, the secondarycleansing method includes applying heat, sonication, positive pressure,negative pressure, or a combination thereof. In some embodiments, themarine organism skeletal derivative-based solid material furthercomprises a bone filler, bone cement, bioglass or bone substitutematerial.

In some embodiments, the process further comprises the step ofcontacting said marine organism skeletal derivative solid material witha fluid for from 0.1-15 minutes, or in some embodiments, from 1-2seconds to 20 minutes, or in some embodiments from 0.5-40 minutes, or insome embodiments from 0.1-60 minutes, allowing for spontaneous fluiduptake of said fluid within said marine organism skeletalderivative-based solid material to arrive at said spontaneous fluiduptake value. In some embodiments, the process further comprises thestep of contacting said marine organism skeletal derivative-based solidmaterial with a fluid for from 12 up to 24 hours, or in someembodiments, for from 2 to up to 15 hours, or in some embodiments, forfrom 1 to up to 24 hours, or in some embodiments from 0.05 up to 24hours, or in some embodiments, for from 6 to up to 24 hours, or in someembodiments, for from 18 to up to 24 hours, allowing for spontaneousfluid uptake of said fluid within said marine organism skeletalderivative-based solid material to arrive at said spontaneous fluiduptake value.

In some embodiments, the process further comprises the step ofcontacting said marine organism skeletal derivative-based solid materialwith a fluid and applying negative pressure to said marine organismskeletal derivative-based solid material to provide for maximal uptakeof said fluid within said marine organism skeletal derivative-basedsolid material to arrive at said total fluid uptake value. In someembodiments, the specific fluid uptake capacity value is a function ofchange in weight in said marine organism skeletal derivative-based solidmaterial. In some embodiments, the specific fluid uptake capacity valueis a function of change in fluid volume of applied fluid to said marineorganism skeletal derivative-based solid material. In some embodiments,the fluid is a protein-containing, salt-containing or carbohydratecontaining solution. In some embodiments, the biologic fluid isautologous with respect to a cell or tissue of a subject when said solidsubstrate is contacted with a cell or tissue of said subject. In someembodiments, the fluid is water. In some embodiments, the marineorganism skeletal derivative-based solid material is isolated from aPorites species, Goniopora, Millepora species or an Acropora species. Insome embodiments, the solid substrate is isolated from a barnacle ormollusk, or bone or ivory or dentin. In some embodiments, the solidsubstrate is comprised of nacre.

In some embodiments, the marine organism skeletal derivative-based solidmaterial approximates the form of a cylinder, cone, tac, pin, screw,rectangular bar, plate, disc, pyramid, granule, powder, coral sand,ball, bone, condyle, rib, vertebra or cube. In some embodiments, themarine organism skeletal derivative-based solid material approximates ashape which accommodates a site of desired tissue growth or repair. Insome embodiments, the marine organism skeletal derivative-based solidmaterial comprises a hollow or hollows along a Cartesian coordinate axisof said coralline-based solid material.

In some embodiments, this invention provides a process for selection ofan optimized marine organism skeletal derivative-based solid substratefor promoting cell or tissue growth or restored function, said processcomprising:

-   -   Isolating or preparing a marine organism skeletal        derivative-based solid material;    -   establishing a specific fluid uptake capacity value of said        marine organism skeletal derivative-based solid material, which        specific fluid uptake capacity value is determined by        establishing a spontaneous fluid uptake value divided by a total        fluid uptake value; or establishing a contact angle value; and    -   selecting a marine organism skeletal derivative-based solid        material characterized by a specific fluid uptake capacity value        of at least 75% or characterized by having a contact angle value        of less than 60 degrees, when in contact with a fluid    -   establishing the presence of a substantially rough surface on        said marine organism skeletal derivative-based solid material,        which substantially rough surface is determined by scanning        electron microscopy, x-ray diffraction or atomic force        microscopy; and    -   selecting a marine organism skeletal derivative-based solid        material characterized by a determination of the presence of a        substantially rough surface on said marine organism skeletal        derivative-based solid material.

In some embodiments, this invention provides a process for selection ofan optimized marine organism skeletal derivative-based solid substratefor promoting cell or tissue growth or restored function, said processcomprising:

-   -   Isolating or preparing a marine organism skeletal        derivative-based solid material;    -   establishing a specific fluid uptake capacity value of said        marine organism skeletal derivative-based solid material, which        specific fluid uptake capacity value is determined by        establishing a spontaneous fluid uptake value divided by a total        fluid uptake value; or establishing a contact angle value;    -   selecting a marine organism skeletal derivative-based solid        material characterized by a specific fluid uptake capacity value        of at least 75% or characterized by having a contact angle value        of less than 60 degrees, when in contact with a fluid; and    -   establishing a crystalline composition or structure of said        marine organism skeletal derivative-based solid material, by use        of x-ray diffraction or Feigl stain positive staining.

All publications, patents, and patent applications mentioned herein arehereby incorporated by reference in their entirety as if each individualpublication or patent was specifically and individually indicated to beincorporated by reference. In case of a conflict between thespecification and an incorporated reference, the specification shallcontrol. Where number ranges are given in this document, endpoints areincluded within the range. Furthermore, it is to be understood thatunless otherwise indicated or otherwise evident from the context andunderstanding of one of ordinary skill in the art, values that areexpressed as ranges can assume any specific value or sub-range withinthe stated ranges, optionally including or excluding either or bothendpoints, in different embodiments of the invention, to the tenth ofthe unit of the lower limit of the range, unless the context clearlydictates otherwise. Where a percentage is recited in reference to avalue that intrinsically has units that are whole numbers, any resultingfraction may be rounded to the nearest whole number.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F provide a series of photographs of implants, which wereassessed for their ability to imbibe a biologic fluid, in this case,whole human blood. FIGS. 1A-1C show 3 types of patterns of uptake withinsmall coral solid substrate samples, reasonably full uptake asdetermined by surface color change observation, moderate uptake andminimal uptake, respectively. FIGS. 1D-1F show a larger block of thecoral solid substrate from which the smaller implants were isolated.

FIG. 2 presents a flow chart for an embodied screening protocol for theidentification of optimized marine organism skeletal derivative-basedsolid substrates for promoting cell or tissue growth or restoredfunction.

FIGS. 3A-3F demonstrate the correlation between biologic fluid uptakebefore implantation and site healing over time. Implantation sitestreated with implants characterized by significant water and blooduptake, or minimal uptake thereof within the implant beforeimplantation, were evaluated macroscopically, at 4 weeks post. Tissueconsistent with cartilage appearance substantially covered the implant,in samples with significant fluid uptake, whereas samples which werecharacterized by minimal/diminished fluid uptake presented with a morefibrous covering over the implant implantation (FIG. 3A versus 3D,respectively). X-ray and micro-CT analysis of the respective implantscharacterized by minimal/diminished fluid uptake [FIGS. 3B and 3C]versus those characterized by significant fluid uptake [FIGS. 3E and 3F]demonstrated that implant characterized by significant fluid uptake areappeared to be properly integrated within the implantation site with nosignificant adverse reaction with excellent osteointegration,ostoconduction and osteotransduction, while implants characterized byminimal/diminished fluid uptake are appeared to induce bone resorption,lysis and loss of mechanical integrity, possibly due to enhancedosteoclast activity.

FIGS. 4A-4D provide photographs of embodied implants evaluated for theircontact angle, when exposed to fluid. FIGS. 4A and 4B show where thesamples which were assessed for their contact angle characterizationwere cut from a larger block, and FIGS. 4C and 4D provide the contactangle values obtained for the indicated regions. The majority of regionsof the block assessed in FIGS. 4A and 4B provided for a contact angleprimarily of less than 60 degrees. Certain areas in FIGS. 4C and 4Dprovided for a contact angle of between 60 and 90 degrees (FIG. 4C) andover 90 degrees (FIG. 4D).

FIGS. 5A-5C are photographs of embodied implants evaluated for theircontact angle, when exposed to fluid. FIG. 5A shows regions cut from thelarger block, which were assessed for their contact anglecharacterization. The majority of regions of the block assessed in FIGS.5B and 5C provided for a contact angle primarily of less than 60degrees. Certain areas in FIGS. 5B and 5C provided for a contact angleof between 60 and 90 degrees and over 90 degrees.

FIGS. 6A and 6B similarly provide photographs of embodied implantsevaluated for their contact angle, when exposed to fluid. FIG. 6A showsregions cut from the larger block, which were assessed for their contactangle characterization. The majority of regions of the block assessed inFIG. 6B provided for a contact angle primarily of less than 60 degrees.Certain areas in FIG. 6B provided for a contact angle of between 60 and90 degrees and over 90 degrees.

FIGS. 7A-7E demonstrate the results of ESEM analysis showing comparativesurface wetting characteristics. The sample assessed in FIG. 7A showed azero drop angle value, and no drop formation, indicating highhydrophilic structure. FIG. 7B depicts a sample, which following theapplication of fluid, failed to “wet” when water was applied. FIG. 7Cshows that following re-desiccation, water droplets were evident on thesurface, consistent with a phenotype of poor surface wetting. FIG. 7Dpresent the results for a different sample, with results consistent withof contact angle less than 60 degrees and FIG. 7E present the resultsfor a different sample, with contact angle higher than 60 degrees.

FIGS. 8A-8F demonstrate the microscopic structure as determined by ESEM,of isolated substrates characterized by minimal biologic fluid uptake(FIGS. 8A-8C), versus those characterized by substantial biologic fluiduptake (FIGS. 8D-8F) at various magnifications in samples with minimalbiologic fluid uptake indicate a much smoother external surface ascompared to samples with substantial uptake (FIGS. 8A-8C versus 8D-8F).

FIGS. 9A-9F demonstrate the microscopic structure as determined by AFM,of isolated substrates characterized by minimal biologic fluid uptake,versus those characterized by substantial biologic fluid uptake atvarious magnifications in samples with minimal biologic fluid uptakeindicate a much smoother external surface as compared to samples withsubstantial uptake (FIGS. 9A-9C versus 9D-9F).

FIG. 10 plots the SFUC value as a function of coral sample assessed,prior to and following application of Tween 80 to such samples.

FIG. 11 plots the SFUC value as a function of coral sample assessed,prior to and following application of Tween 80 and sonication to suchsamples.

FIG. 12 plots the SFUC value as a function of coral sample assessed,prior to and following application of pluronic with or without Tween 80to such samples

FIG. 13 plots the SFUC value as a function of coral sample assessed,prior to and following application of absolute ethanol to such samples

FIG. 14 plots the SFUC value as a function of coral sample assessed,prior to and following application of methylene blue to such samples.

FIG. 15 plots the SFUC value as a function of coral sample assessed,prior to and following application of hyaluronic acid to such samples.

FIG. 16 plots the SFUC value as a function of coral sample assessed,prior to and following application of chondroitin sulfate to suchsamples.

FIG. 17 plots the SWC values of implants from two samples (a firstevaluating (n=13) and the second evaluating (n=15) corals) successivelyexposed to purification chemicals: hypochlorite and hydrogen peroxideand followed by an absolute ethanol extraction. *P<0.05; **P<0.01.

FIGS. 18 and 19 similarly plot the average SWC values for implantsexposed to the purification step and following an ethanol extractionstep.

FIGS. 20A and 20B schematically depict an embodied automated apparatusof this invention in side and top view.

FIGS. 21A-21C present scanning electron micrographs of cell adhesion tocoral substrates having a low specific fluid uptake capacity value (FIG.21A and FIG. 21B) versus a high specific fluid uptake capacity value(FIG. 21C).

FIGS. 22A-22B graphically depict the HEPM cell proliferation andviability values as determined by alamarBlue® assay in samples having alow versus high specific fluid uptake capacity value.

FIGS. 23A-23F depict traditional Feigl staining as reported in theliterature (FIG. 23A-23B), and blood uptake versus Feigl staining incoral samples isolated and processed by an embodied process of thisinvention, before (FIG. 23C, 23D) and following a further ethanolpurification step (FIG. 23E, 23F).

DETAILED DESCRIPTION OF THE PRESENT INVENTION

This invention provides, inter alia, processes for selecting for andobtaining optimized solid substrates for promoting cell or tissue growthor restored function and materials obtained thereby.

In some embodiments, the invention provides a solid substrate forpromoting cell or tissue growth or restored function, which solidsubstrate comprises coral and is characterized by a specific fluiduptake capacity value of at least 75%, which specific fluid uptakecapacity value is determined by establishing a spontaneous fluid uptakevalue divided by a total fluid uptake value.

In some embodiments, this invention provides a solid substrate forpromoting cell or tissue growth or restored function, which solidsubstrate comprises a organism skeletal derivative and is characterizedby having a contact angle value of less than 60 degrees, when in contactwith a fluid.

In some embodiments, the invention provides a solid substrate forpromoting cell or tissue growth or restored function, which solidsubstrate comprises a marine organism skeletal derivative and ischaracterized by substantial surface roughness (Ra) as measured byscanning electron microscopy or atomic force microscopy.

The solid substrates of this invention will comprise a marine organismskeletal derivative-based material.

In some embodiments, the term “marine organism skeletal derivative-basedmaterial” refers to a solid piece or ground material derived from amarine organism, and from a skeletal component of the organism, such asan exoskeleton of the same further processed to be suitable forimplantation in a human or veterinary subject, and further processed tobe optimized for implantation as described hereinunder.

In some embodiments, the term “marine organism skeletal derivative-basedmaterial” refers to a coralline-based material further processed to besuitable for implantation in a human or veterinary subject, and furtherprocessed to be optimized for implantation as described hereinunder.

Coral, which is mainly comprised of CaCO₃ has been shown to possess theadvantage of supporting fast cellular invasion, adherence andproliferation. Coral has been shown to be an effective substrate forfacilitation of the adherence, proliferation and differentiation ofmesenchymal stem cells, and ultimate incorporation into cartilage and/orbone tissue. Coral has also been shown to serve as an excellentsubstrate for promoting adherence and proliferation of a number of othercell types, serving as an excellent support for cell and tissue growth.

The terms “coral” and “calcium carbonate” and “aragonite” and “calcite”may be used interchangeably herein, unless specifically stated to thecontrary.

In some embodiments, the term “marine organism skeletal derivative-basedmaterial” refers to a coral or coral derivative further processed to besuitable for implantation in a human or veterinary subject, and furtherprocessed to be optimized for implantation as described hereinunder. Insome embodiments, the term “marine organism skeletal derivative-basedmaterial” refers to barnacle or mollusk-derived skeletal materialfurther processed to be suitable for implantation in a human orveterinary subject, and further processed to be optimized forimplantation as described hereinunder, and in some embodiments,inclusion of nacre further processed to be suitable for implantation ina human or veterinary subject, and further processed to be optimized forimplantation as described hereinunder is contemplated.

In some embodiments, the term “marine organism skeletal derivative-basedmaterial” refers to a coral or coral derivative, which is isolated froma native marine organism, and subsequently processed as describedherein, so as to be suitable for implantation within a human orveterinary subject, which marine organism skeletal derivative-basedmaterial has been specifically subjected to further processing, asdescribed herein, including a prior cleaning and purification step, inorder to convert a suboptimal isolated marine organism skeletalderivative-based material to an optimized marine organism skeletalderivative-based material for promoting cell or tissue growth orrestored function.

In some embodiments, such optimization specifically includes contactingsaid marine organism skeletal derivative-based solid material with anamphiphilic material, a polar solvent, a cationic material, an anionicmaterial, or a combination thereof.

In some embodiments, the solid substrate contains ground particlesderived from coral, suspended in a biocompatible matrix. In someembodiments, the biocompatible matrix is a hydrogel.

In some embodiments, reference to an “implant” or “plug” or “solidsubstrate”, as used herein refers to any embodiment or combinedembodiments as herein described with regard to the solid substrates andto be considered as being included in the described aspect of thisinvention. For example, reference to a “solid substrate” as used herein,is to be understood to refer to any embodiment of a solid substrate asdescribed herein being applicable for the indicated purpose orcontaining the indicated attribute, etc.

In one embodiment, “solid substrate” refers to a shaped platform usedfor cell and/or tissue repair and/or restored function, wherein theshaped platform provides a site for such repair and/or restoredfunction. In one embodiment, the solid substrate is a temporaryplatform. In one embodiment, “temporary platform” refers to a naturaldegradation of a coral of this invention that occurs over time duringsuch repair, wherein the natural fully or partially degradation of thecoral may results in a change of solid substrate shape over time and/orchange in solid substrate size over time.

It will be appreciated that different species of coral vary in terms oftheir average pore diameter and pore volume and the inventioncontemplates use of any such coral as a starting material for thepreparation of the solid substrates as herein described, where the solidsubstrate is characterized in that it is characterized by a specificfluid uptake capacity value of at least 75% further processed to besuitable for implantation in a human or veterinary subject, and furtherprocessed to be optimized for implantation as described hereinunder.

As used herein, the term “pore volume” refers to volume or open spacesinside the porous scaffolding of this invention. Pore volume isdetermined by any means known in the art. Porosity can be calculated bystandard methods, an example of which is provided further hereinbelow,see for example, Karageorgiou V, Kaplan D. (2005) “Porosity of 3Dbiomaterial scaffolds and osteogenesis” Biomaterials; 26(27):5474-91,which is hereby incorporated by reference in its entirety.

It will be appreciated that the term “coral” will refer to a startingmaterial from which aragonite, calcium carbonate, calcite, orhydroxyapatite etc. may be isolated and further processed to be suitablefor implantation in a human or veterinary subject, and further processedto be optimized for implantation as described hereinunder.

In one embodiment, the solid substrates, processes and/or kits of thisinvention employ use of a coral further processed to be suitable forimplantation in a human or veterinary subject, and further processed tobe optimized for implantation as described hereinunder. In oneembodiment, the coral comprise any species, including, inter alia,Porites, Acropora, Goniopora, Millepora, or a combination thereof. Inanother embodiment the solid substrates, processes and/or kits of thisinvention employ use of nacre, mollusc shell, or bone morsels.

In one embodiment, the coral is from the Porites species. In oneembodiment, the coral is Porites Lutea. In one embodiment, the coral isfrom the Acropora species. In one embodiment, the coral is Acroporagrandis, which in one embodiment is very common, fast growing, and easyto grow in culture. Thus, in one embodiment Acropora samples can beeasily collected in sheltered areas of the coral reefs and collectionfrom the coral reefs can be avoided by use of cultured coral material.

In another embodiment, the coral is from the Millepora species. In oneembodiment, the coral is Millepora dichotoma. In one embodiment, thecoral has a pore size of 150 μm and can be cloned and cultured, makingMillerpora useful as a framework in the solid substrates, methods and/orkits of this invention.

In one embodiment, the coral is from the Goniopora species. In someembodiments, the coral is Goniopora albiconus, Goniopora burgosi,Goniopora cellulosa, Goniopora ceylon, Goniopora ciliatus, Gonioporacolumna, Goniopora djiboutiensis, Goniopora eclipsensis, Gonioporafruticosa, Goniopora gracilis, Goniopora klunzingeri, Goniopora lobata,Goniopora mauritiensis, Goniopora minor, Goniopora norfolkensis,Goniopora palmensis, Goniopora pandoraensis, Goniopora parvistella,Goniopora pearsoni, Goniopora pendulus, Goniopora planulata, Gonioporapolyformis, Goniopora reptans, Goniopora savignyi, Gonioporasomaliensis, Goniopora stokes, Goniopora stutchburyi, Goniopora sultani,Goniopora tenella, Goniopora tenuidens or Goniopora viridis.

In another embodiment, the coral is from any one or more of thefollowing species Favites halicora; Goniastrea retiformis; Acanthastreaechinata; Acanthastrea hemprichi; Acanthastrea ishigakiensis; Acroporaaspera; Acropora austera; Acropora sp. “brown digitate”; Acroporacarduus; Acropora cerealis; Acropora chesterfieldensis; Acroporaclathrata; Acropora cophodactyla; Acropora sp. “danai-like”; Acroporadivaricata; Acropora donei; Acropora echinata; Acropora efflorescens;Acropora gemmifera; Acropora globiceps; Acropora granulosa; Acropora cfhemprichi; Acropora kosurini; Acropora cf loisettae; Acroporalongicyathus; Acropora loripes; Acropora cf lutkeni; Acroporapaniculata; Acropora proximalis; Acropora rudis; Acropora selago;Acropora solitaryensis; Acropora cf spicifera as per Veron; Acropora cfspicifera as per Wallace; Acropora tenuis; Acropora valenciennesi;Acropora vaughani; Acropora vermiculata; Astreopora gracilis; Astreoporamyriophthalma; Astreopora randalli; Astreopora suggesta; Australomussarowleyensis; Coscinaraea collumna; Coscinaraea crassa; Cynarinalacrymalis; Distichopora violacea; Echinophyllia echinata; Echinophylliacf echinoporoides; Echinopora gemmacea; Echinopora hirsutissima;Euphyllia ancora; Euphyllia divisa; Euphyllia yaeyamensis; Faviarotundata; Favia truncatus; Favites acuticollis; Favities pentagona;Fungia granulosa; Fungia klunzingeri; Fungia mollucensis; Galaxeaacrhelia; Goniastrea edwardsi; Goniastea minuta; Hydnophora pilosa;Leptoseris explanata; Leptoseris incrustans; Leptoseris mycetoseroides;Leptoseris scabra; Leptoseris yabei; Lithophyllon undulatum; Lobophylliahemprichii; Merulina scabricula; Millepora dichotoma; Millepora exaesa;Millipora intricata; Millepora murrayensis; Millipora platyphylla;Monastrea curta; Monastrea colemani; Montipora caliculata; Montiporacapitata; Montipora foveolata; Montipora meandrina; Montiporatuberculosa; Montipora cf vietnamensis; Oulophyllia laevis; Oxyporacrassispinosa; Oxypora lacera; Pavona bipartita; Pavona venosa; Pectiniaalcicornis; Pectinia paeonea; Platygyra acuta; Platygyra pini; Platygyrasp “green”; Platygyra verweyi; Podabacia cf lanakensis; Porites annae;Porites cylindrica; Porites evermanni; Porites monticulosa; Psammocoradigitata; Psammocora explanulata; Psammocora haimeana; Psammocorasuperficialis; Sandalolitha dentata; Seriatopora caliendrum;Stylocoeniella armata; Stylocoeniella guentheri; Stylaster sp.; Tubiporamusica; Turbinaria stellulata; or any coral known in the art, or acombination thereof.

In another embodiment, derivatives of marine animals—such as coral,sponges, moluscs shells and other related organisms may be used in thesolid substrates, methods and/or kits of this invention may beMadreporaria, Helioporida of the order Coenothecalia, Tubipora of theorder Stolonifera, Millepora of the order Milleporina, or others knownin the art. In some embodiments, coral for use in the substrates,methods and/or kits of this invention may comprise scleractinian coral,including in some embodiments, Goniopora and others. In someembodiments, coral for use in the substrates, methods and/or kits ofthis invention may comprise Alveoppora. In some embodiments, coral foruse in the substrates, methods and/or kits of this invention maycomprise bamboo corals, including in some embodiments, coral from thefamily Isididae, genera Keratoisis, Isidella, and others.

In one embodiment of this invention, the term “coral” refers to coralwhich is cut from a single piece of coral and further processed to besuitable for implantation in a human or veterinary subject, and stillfurther processed to be optimized for implantation as describedhereinunder.

In some embodiments, the solid substrate is of any desired shape.

In one embodiment, coral may be machined into a variety ofconfigurations, and quite complex shapes such as cylindrical structuresand threaded structures may be formed by appropriate machine or otherprocessing, such as chemical processing. In another embodiment, coralmay be shaped to form solid blocks, rods or granular forms. In oneembodiment, coralline materials are shaped in such a way as to conformto the shape of a desired tissue structure or to fill gap and contourdefects in a potential implantation site. In one embodiment, coral isimplanted in an orientation that allows it to contact the maximumsurface area of an adjacent-located tissue structure.

In some embodiments, the solid substrate approximates the form of acylinder, cone, tac, pin, screw, rectangular bar, plate, disc, pyramid,granule, powder, coral sand, ball, bone, condyle, rib, vertebra, orcube. In some embodiments, the solid substrate approximates a shapewhich accommodates a site of desired tissue growth or repair.

In some embodiments, the solid substrate comprises a hollow or hollowsalong a Cartesian coordinate axis of said solid substrate.

In one embodiment, the size of coral solid substrates may be any sizethat would be useful for the purposes of the present invention, as wouldbe known to one of skill in the Art depending on the purpose. Forexample and in one embodiment, the solid substrate may be substantiallythe same size as the structure it is meant to replace, while in anotherembodiment, the solid substrate or a portion thereof may be the size ofa defect, fissure or fracture such that it may be placed therein toenhance/replace tissue formation/function in a discrete location. In oneembodiment, a coral for use in a solid substrate of this inventioncomprises an average void diameter, average pore size or a combinationthereof appropriate for cell seeding and/or development of vasculature.

The processes and materials of this invention, in some embodiments, relyon the isolation or preparation of the marine organism skeletalderivative-based solid material for use. In some embodiments, suchisolation and preparation may include, inter alia, selection of adesired sample, including selecting a number of samples from a desiredregion of a growth ring in a larger coral sample, and/or in someembodiments, first processing of such sample, and/or in someembodiments, prescreening of such samples for their uptake of a fluid asdescribed herein, and/or in some embodiments, further processing of suchsample, as herein described. According to this aspect, such isolationand preparation will be effected prior to the described establishment ofa specific fluid uptake capacity value of said marine organism skeletalderivative-based solid material, as herein described, and in someembodiments, prior to the selection of the marine organism skeletalderivative-based solid material as characterized by the describedspecific fluid uptake capacity value of at least 75% or contact anglevalue of less than 60 degrees, when in contact with a fluid, or surfaceroughness or Feigl stain positivity as herein described.

In one embodiment, coral is washed, bleached, frozen, dried, exposed toelectrical forces, magnetic forces or ultrasound waves or microwaves orelectromagnetic radiation or high pressure or a combination thereofprior to use thereof. According to this aspect, and in some embodiments,the coral is exposed to further processing, as described hereinunder.

In some embodiments, the solid substrate is of a size that isappropriate for the intended purpose, as will be appreciated by theskilled artisan.

For example, and in some embodiments, solid substrates for use inosteochondral therapy or repair may make use of a substrate that iscylindrical or oval in shape and in some embodiments, solid substratesof this invention and/or for use in the kits and methods as describedmay have a diameter of about 5-15 mm, and a height of about 5-25 mm. Insome embodiments, the solid substrate has a diameter of about 1-35 mm,and a height of about 1-45 mm, or about 5-40 mm, and a height of about5-60 mm, or about 5-15 mm, and a height of about 5-45 mm. 5-30 mm, 15-60mm, or larger.

For example, and in some embodiments, solid substrates of this inventionand/or for use in the kits and methods as described may make use of asubstrate that is cylindrical or oval in shape and in some embodiments,solid substrates of this invention and/or for use in the kits andmethods as described may have a diameter in the nanometer or micrometerscale. In some embodiments, solid substrates for use in osteochondraltherapy or repair may make use of a substrate that is cylindrical oroval in shape and has a diameter of about 1-100 nm, or in someembodiments, having a diameter of about 50-1000 nm, or in someembodiments, having a diameter of about 10-2000 nm, or in someembodiments, having a diameter of about 100-4000 nm. In someembodiments, solid substrates for use in osteochondral therapy or repairmay make use of a substrate that is cylindrical or oval in shape and hasa diameter of about 1-100 μm, or in some embodiments, having a diameterof about 50-1000 μm, or in some embodiments, having a diameter of about10-2000 μm, or in some embodiments, having a diameter of about 100-4000μm.

For example, and in some embodiments, solid substrates of this inventionand/or for use in the kits and methods as described may make use of asubstrate that is cylindrical or oval in shape and in some embodiments,solid substrates of this invention and/or for use in the kits andmethods as described may have a diameter in the millimeter or centimeterscale. In some embodiments, solid substrates for use in osteochondraltherapy or repair may make use of a substrate that is cylindrical oroval in shape and has a diameter of about 1-100 mm, or in someembodiments, having a diameter of about 50-1000 mm, or in someembodiments, having a diameter of about 10-2000 mm, or in someembodiments, having a diameter of about 100-4000 mm. In someembodiments, solid substrates for use in osteochondral therapy or repairmay make use of a substrate that is cylindrical or oval in shape and hasa diameter of about 1-100 cm, or in some embodiments, having a diameterof about 50-1000 cm, or in some embodiments, having a diameter of about10-2000 cm, or in some embodiments, having a diameter of about 100-4000cm.

It will be appreciated by the skilled artisan that the size of thesubstrate may be so selected so as to be suitable to a particularapplication, for example, when using as a scaffolding material for bonerepair, then the size may approximate the dimensions of a long bone inthe subject. Accordingly, this invention is not to be limited by thesize of the solid substrate.

The average diameter of the voids within the phases of the solidsubstrates of this invention may be determined by any means, includingdigital images analysis.

In some embodiments, the coral for use in accordance with the instantinvention may be prepared as described in PCT International Applicationpublication Number WO 2009/066283, PCT International Applicationpublication Number WO 2010/058400, PCT International Applicationpublication Number WO 2010/146574 and PCT International Applicationpublication Number WO 2010/146574, each of which is fully incorporatedby reference herein, in its entirety.

In some embodiments, coral is isolated from a natural source by knownmethods, and as described herein. In some embodiments, care is taken toisolate coral slices from a region of one or more growth rings within alarger coral sample, which region has been shown to possess theappropriate specific fluid uptake capacity value, the appropriatecontact angle value and/or surface roughness, as described herein and insome embodiments, is then exposed to further processing as describedhereinunder.

The processes of this invention promote obtaining a solid substrate ofthis invention, characterized by a specific fluid uptake capacity valueas desired for the specific application for example of at least 75%,which specific fluid uptake capacity value is determined by establishinga spontaneous fluid uptake value divided by a total fluid uptake value.

As described and exemplified herein, for example, as described inExamples 3 and 4, a specific fluid uptake capacity value may bedetermined by evaluating spontaneous uptake of a biologic fluid versus atotal uptake capacity for a given sample and arriving at the specificfluid uptake capacity level, whereby if the value is over 75%, then suchsolid substrate will be used in applications promoting cell and tissuegrowth and/or restored function.

In some embodiments, the process for selection of the solid substratecomprises isolating a sample of a coralline-based solid material andestablishing a specific fluid uptake capacity value of the material,which specific fluid uptake capacity value is determined by establishinga spontaneous fluid uptake value divided by a total fluid uptake valueand selecting material characterized by a specific fluid uptake capacityvalue of at least 75%.

In some embodiments, the biologic fluid is blood, and in someembodiments, the biologic fluid is water. In some embodiments, thebiologic fluid is hydrophilic.

In some embodiments, the biologic fluid is autologous with respect to acell or tissue of a subject when said solid substrate is contacted withsuch cell or tissue of said subject.

It will be understood that the biologic fluid may be any fluid which isbiocompatible and whose incorporation is appropriate within a solidsubstrate for the desired application.

In some embodiments, the process further comprises the step ofcontacting the material with a fluid for from 2-15 minutes to promotespontaneous fluid uptake of said fluid within said coralline-based solidmaterial to arrive at said spontaneous fluid uptake value. In someembodiments, the process may allow for the contacting of the materialwith a fluid for from 0.5-15 minutes, or in some embodiments, from 0.5-5minutes, or in some embodiments, 10-60 minutes, or in some embodiments,from 60 to 90 minutes, or in some embodiments, other intervals, topromote spontaneous fluid uptake. The skilled artisan will appreciatethat the amount of time for which the fluid is applied to determine thespontaneous uptake may be extended or shortened as a function of thedimensions and geometry of the sample substrate being assessed. In someembodiments, when a larger sample is being assessed, the process furthercomprises the step of contacting the material with a fluid for from 2-24hours to promote spontaneous fluid uptake of said fluid within saidcoralline-based solid material to arrive at said spontaneous fluiduptake value

In some embodiments, the process further comprises the step ofcontacting said marine organism skeletal derivative-based solid materialwith a fluid and applying negative pressure, or in some embodiments,mechanical pressure, to said coralline-based solid material to promotemaximal uptake of said fluid within said coralline-based solid materialto arrive at said total fluid uptake value. In some embodiments,application of positive pressure is via the application of a vacuum orin some embodiments, mechanical pressure, to the substrate immersed inthe fluid, promoting entry of the fluid therewithin.

In some embodiments, the process may further comprise the step ofcontacting said coralline-based solid material with a fluid and applyingpositive pressure to said coralline-based solid material to promotemaximal uptake of said fluid within said coralline-based solid materialto arrive at said total fluid uptake value. According to this aspect,and in some embodiments, care will be taken to ensure that theapplication of pressure does not in any way compromise the structuralintegrity of the solid substrate.

In some embodiments, application of positive pressure is via any manualmeans, for example, via the use of any applicator, syringe, etc.,gravitational pressure, and others, as will be appreciated by theskilled artisan. In some embodiments, application of positive pressureis via forced osmosis, centrifugation and others. In some embodiments,combinations of the described methods and others are envisioned.

In some embodiments, a prescreening step may be undertaken. For example,and in some embodiments, a coral slice of a desired thickness is taken,which for example may be perpendicular to the coral sample ring growth.The slice may be evaluated for rapid uptake of a biological fluid, suchas, for example, uptake of a colored proteinaceous fluid, such as blood.In some embodiments, blood from any source may be used, such as, forexample, from livestock or other sources.

Samples which provide a rapid uptake as part of the described prescreenprocedure may be further assessed for their specific fluid uptakecapacity value.

For example, and in some embodiments, smaller samples or specificscaffolds may be isolated from the block from which the coral slice wastaken for prescreening, from regions which were determined by theprescreen to provide rapid uptake of the biological fluid.

In some embodiments, scaffold and/or smaller samples are dried and thensubjected to further processing. Such further processing, for example,ensures removal of matter, which would render the implants unfit forimplantation in human or veterinary subjects. In some embodiments, suchprocessing produces a product that is fit for implantation, inaccordance with any regulatory body guidance, such as, for example, theASTM F 1185-03: Standard Specification for Composition ofHydroxylapatite for Surgical Implants, or ASTM F 1581-08: StandardSpecification for Composition of Anorganic Bone for Surgical Implants.

In some embodiments, such further processing includes the oxidation oforganic residuals in the scaffold and/or smaller samples, and subsequentelimination of the oxidizing agent used. In some aspects, such oxidizingagent may include sodium hypochlorite, hydrogen peroxide (solutionsthereof) or use of both, which in some embodiments, is followed by theapplication of a polar solvent.

In some embodiments, such further processing steps may be undertakenfollowing the establishment of a specific fluid uptake capacity valueand in some embodiments, such further processing steps may be undertakenprior to establishing the specific fluid uptake capacity value for thesample(s).

According to this aspect, and in some embodiments, such scaffold orsmaller samples may be fully dried, and then assessed for theirspontaneous fluid uptake value, for example, as described in Example 1below. For example, the dry sample may be immersed in water and thespontaneous fluid uptake value assessed, followed by an assessment ofthe total fluid uptake value. According to this aspect, and in oneembodiment, samples producing a specific fluid uptake capacity value ofat least 75% are selected for further processing. In some embodiments,samples producing a specific fluid uptake capacity value of at least60-95% are selected for still further processing.

In some aspects, such still further processing includes a process toimprove or further optimize a specific fluid uptake capacity value of agiven scaffold and/or sample, including processes to optimize same asdescribed herein. In some embodiments, such further processing mayinclude subjecting the scaffold and/or sample to polar solvent exposure,as herein described.

In some embodiments, the invention also provides a process forconverting a suboptimal marine organism skeletal derivative-based solidsubstrate to an optimized marine organism skeletal derivative-basedsolid substrates for promoting cell or tissue growth or restoredfunction, said process comprising:

-   -   a) establishing a specific fluid uptake capacity value for a        group of marine organism skeletal derivative-based solid        materials, which specific fluid uptake capacity value is        determined by establishing a spontaneous fluid uptake value        divided by a total fluid uptake value for each sample in said        group;    -   b) selecting a marine organism skeletal derivative-based solid        material characterized by a specific fluid uptake capacity        value;    -   c) subjecting said marine organism skeletal derivative-based        solid material of (b) to cold plasma treatment, corona        treatment, or a combination thereof;    -   d) determining a specific fluid uptake capacity as in (a) in        said marine organism skeletal derivative-based solid materials        obtained in (c); and    -   e) selecting marine organism skeletal derivative-based solid        materials obtained in (d) having a newly established increased        specific fluid uptake capacity value.

In some embodiments, the further processing specifically contemplatescold plasma treatment, corona treatment or a combination thereof. Insome embodiments, the further processing may include surface conversionto hydroxyapatite, for example, via hydrothermal reaction, by knownmethods.

In some embodiments, the solid substrates for promoting cell or tissuegrowth or restored function of this invention comprise an organismskeletal derivative characterized by having a contact angle value ofless than 60 degrees, when in contact with a fluid.

Example 5 demonstrated that solid substrates characterized by a contactangle value of less than 60 degrees is comparable to samples having aspecific fluid uptake capacity value of at least 75%, and therefore suchsamples are also to be considered as comprising part of this invention.

Methods for determining a contact angle are well known, and anyappropriate method can be used. One embodiment of such a method isprovided herein with regard to Example 5 and as described hereinabove.

In some embodiments the structure composition of the coral or coralderivative is determined by X-ray diffraction (XRD) or Feigl solutionpositive staining.

In some embodiments, the term “Feigl solution positive staining” or“Feigl stain positivity” refers to a staining pattern consistent with astandard Feigl staining pattern (black) as known in the art and asdescribed herein, indicative of an aragonite crystalline structure.

Similarly, in some embodiments of this invention, there is provided aprocess for selection of an optimized organism skeletal derivative-basedsolid substrate for promoting cell or tissue growth or restoredfunction, said process comprising:

-   -   Isolating or preparing a organism skeletal derivative-based        solid material;    -   contacting said organism skeletal derivative-based solid        material with a fluid and establishing a contact angle for said        organism skeletal derivative; and    -   selecting a organism skeletal derivative-based solid material        characterized by a contact angle of less than 60 degrees.

In some embodiments, coral-based solid substrates of this invention maybe converted to partially or fully into hydroxyapatite by known methods.

According to this aspect, a solid substrate characterized by a specificfluid uptake capacity value of at least 75%, which specific fluid uptakecapacity value is determined by establishing a spontaneous fluid uptakevalue divided by a total fluid uptake value may be converted tohydroxyapatite, and the indicated activity is present in the convertedsubstrate.

In another embodiment, solid substrate characterized by having a contactangle value of less than 60 degrees, when in contact with a fluid may beconverted to hydroxyapatite, and the indicated activity is present inthe converted substrate.

In another aspect, a solid substrate is converted to hydroxyapatite, andthe same is then assessed for the presence of a specific fluid uptakecapacity value of at least 75%, which specific fluid uptake capacityvalue is determined by establishing a spontaneous fluid uptake valuedivided by a total fluid uptake value, and such substrates fulfillingthe stated criteria are specifically selected and encompassed by thesubject application.

In another aspect, a solid substrate is converted to hydroxyapatite, andthe same is then assessed in terms of its contact angle and whether theangle is less than 60 degrees, and such substrates fulfilling the statedcriteria are specifically selected and encompassed by the subjectapplication.

According to this aspect, and in one embodiment, solid substrates asherein defined, such as, for example, coral samples or nacre or othersas herein described are assessed by selecting a small dry sample for usein the processes as herein described, whose region of isolation from alarger block may be ascertained, in order to provide informationregarding the characteristics of the area in the block from whichadditional samples may be isolated and then used.

In some aspects, the sample is dried under vacuum and/or heated orpressurized or steam treated.

In some embodiments, for aspects relating to a specific fluid uptakecapacity value, such value is a function of change in weight in saidcoralline-based solid material.

According to this aspect and in some embodiments, the dry weight foreach sample is recorded and fluid as described herein is added an assaycontainer.

According to this aspect and in some embodiments, at least 1:1 ratio ofthe size of the sample in mm to the volume of fluid added in ml isapplied to the container. In some embodiments, the amount of fluidapplied is in excess, as compared to the sample size.

According to this aspect and in some embodiments, once the initial fluiduptake is assessed, according to this aspect and in some embodiments,the solid substrate sample is then brought into contact with the fluidand the weight of the solid substrate sample is assessed. In otherembodiments the specific gravity is assessed by gradient centrifugationof by the Archimedean principle.

According to this aspect and in some embodiments, spontaneous fluiduptake is assessed and a spontaneous fluid uptake value is established,based on the change in weight of the sample.

According to this aspect and in some embodiments, the specific fluiduptake capacity value is a function of change in fluid volume of appliedfluid to said marine organism skeletal derivative-based solid material.According to this aspect, spontaneous fluid uptake is assessed and aspontaneous fluid uptake value is established based on the completeuptake of the volume applied to the sample.

According to this aspect and in some embodiments, the process thenfurther comprises contacting a significantly increased amount of fluidwith the sample and applying pressure thereto to promote maximal fluiduptake to the total fluid uptake capacity of the sample.

According to this aspect and in some embodiments, as noted, suchpressure may be either positive or negative pressure, and theapplication time is for a period of time sufficient to ensure maximaluptake of the applied fluid into the marine organism skeletal derivativesample.

According to this aspect and in some embodiments, such time may includean interval of from 0.5-60 minutes, or in some embodiments, when alarger sample is being assessed, such time may include an interval offrom 2-24 hours to arrive at said spontaneous fluid uptake value. Itwill be appreciated that the time intervals recited herein areapplicable for any embodiment with regard thereto as described herein.The skilled artisan will appreciate that the amount of time for whichthe fluid is applied to determine the full capacity fluid uptake may beextended or shortened as a function of the dimensions and geometry ofthe sample substrate being assessed.

According to these aspects, the total fluid uptake capacity is thusassessed and the specific fluid uptake capacity value is thendetermined.

In some embodiments, the invention specifically contemplates solidsubstrates having a specific fluid uptake capacity value exceeding thecutoff value of 75%, for the sample to be noted optimized as a solidsubstrate for promoting cell or tissue growth. It will be appreciatedthat the invention contemplates the stated cutoff value for promoting areasonable value that reduces the presence of appreciable falsepositives, i.e. solid substrates that are not as optimal for the statedapplications.

In some embodiments, the invention specifically contemplates solidsubstrates characterized by having a contact angle value of less than 60degrees, when in contact with a fluid, for the sample to be notedoptimized as a solid substrate for promoting cell or tissue growth. Itwill be appreciated that the invention contemplates the stated cutoffvalue for promoting a reasonable value that reduces the presence ofappreciable false positives, i.e. solid substrates that are not asoptimal for the stated applications.

It is to be noted that the usefulness for coralline substrates forpromoting tissue growth such as cartilage and bone has been previouslyshown. Surprisingly, it has now been found that while numerouscoral-based substrates isolated can be used for such repair, consistentand superior function was found when the substrates were chosenspecifically for their enhanced spontaneous uptake of biologic fluids.Surprisingly, it was found, not only that coral based materials can bean effective material for promoting cell and tissue growth and/orrestored function, but that the spontaneous fluid absorptivecharacteristics of the selected sample of coral for use in the sameprovided even greater activity in this regard.

Without being bound by theory, and representing non-limiting embodimentsof the substrates, processes and applications of this invention,specific selection of marine organism skeletal derivative-based solidsubstrates characterized by the desired specific fluid uptake capacityvalue of at least 75%, or specific selection of organism skeletalderivative-based solid substrates characterized by having a contactangle value of less than 60 degrees, when in contact with a fluid, mayselect for a sample whose vascularization is enhanced, or in someembodiments, whose access to lymph is enhanced, or in some embodiments,whose absorptive capacity heralds an affinity for extracellularmatrix-associated materials, or in some embodiments, whose absorptivecapacity heralds an affinity for cellular attraction includingextravasation from proximal vessels.

In other embodiments, the substrates, processes and applications of thisinvention, specific selection of marine organism skeletalderivative-based solid substrates characterized by the desired specificfluid uptake capacity value of at least 75%, or specific selection oforganism skeletal derivative-based solid substrates characterized byhaving a contact angle value of less than 60 degrees, when in contactwith a fluid, or having a described surface roughness, may select for asample which is particularly useful in anti-cancer applications.

In other embodiments, the substrates, processes and applications of thisinvention, specific selection of marine organism skeletalderivative-based solid substrates characterized by the desired specificfluid uptake capacity value of at least 75%, or specific selection oforganism skeletal derivative-based solid substrates characterized byhaving a contact angle value of less than 60 degrees, or having adescribed surface roughness, when in contact with a fluid, may selectfor a sample which is particularly useful in promotingosteo-integration, osteo-conduction, osteo-transduction, chondrogenesisor cartilage regeneration.

In other embodiments, the substrates, processes and applications of thisinvention, specific selection of marine organism skeletalderivative-based solid substrates characterized by the desired specificfluid uptake capacity value of at least 75%, or specific selection oforganism skeletal derivative-based solid substrates characterized byhaving a contact angle value of less than 60 degrees, when in contactwith a fluid, or having a described surface roughness, may select for asample which is particularly useful in promoting ex-vivothree-dimensional support and structure for cell, tissue or organgrowth. In some embodiments, such cell, tissue or organ growth mayinclude that for heart, muscle, liver, skin, kidney, blood vessel andneuronal growth and development.

In other embodiments, the substrates, processes and applications of thisinvention, specific selection of marine organism skeletalderivative-based solid substrates characterized by the desired specificfluid uptake capacity value of at least 75%, or specific selection oforganism skeletal derivative-based solid substrates characterized byhaving a contact angle value of less than 60 degrees, when in contactwith a fluid, or having a described surface roughness, may select for asample which is particularly useful in promoting ex-vivo or in vitrostem cell growth, proliferation and/or differentiation for applicationsof same, including providing same for three-dimensional support andstructure for cell, tissue or organ growth arising from same, forexample, for applications in heart, muscle, liver, skin, kidney, bloodvessel and neuronal growth and development.

It is to be understood that any of these mechanisms, and others, mayaccount for the phenomenon of enhanced cell or tissue growth or restoredfunction, and that any such mechanism associated with the application ofan optimized marine organism skeletal derivative-based solid substratefor promoting cell or tissue growth or restored function characterizedby a specific fluid uptake capacity value of at least 75% is to beunderstood as being part of this invention.

In some embodiments, samples thus processed and found to becharacterized by a specific fluid uptake capacity value of at least 75%,or specific selection of organism skeletal derivative-based solidsubstrates characterized by having a contact angle value of less than 60degrees, when in contact with a fluid may then be used for the isolationof proximally located regions of a section from which such sample wastaken, which samples can then be reliably used and considered as beingoptimized in accordance with the processes of this invention. In someembodiments, with regard to coral-based samples, such regions mayinclude the entire annual growth ring region within the coral from whichthe sample was derived.

In some embodiments, samples thus processed and found to becharacterized by a specific fluid uptake capacity value of at least 75%,or specific selection of organism skeletal derivative-based solidsubstrates characterized by having a contact angle value of less than 60degrees, when in contact with a fluid, may then be dried fully andutilized for implantation into a subject or for use as an ex-vivosubstrate for cell, tissue or organ growth for subsequent implantation.

In some embodiments, and as exemplified in Example 9, herein, thesubstrates, processes and applications of this invention, whereby amarine organism skeletal derivative-based solid substrates ischaracterized by the desired specific fluid uptake capacity value,contact angle value or surface roughness promotes improved cell adhesionand viability to such substrates. According to this aspect, and in someembodiments, the cell adhesion and cell viability assays demonstratethat samples considered to be optimized for fluid uptake promote greaterfull cell adherence and viability over time.

In some embodiments, when the sample is utilized in vivo in subsequentapplications, in some aspects, the sample is first contacted withautologous biological fluids or materials from the host prior toimplantation into the same, verifying the observed enhanced fluid uptakephenotype as herein described.

A solid substrate of this invention may in some embodiments be insteador concurrently characterized by a substantial surface roughness (Ra) asmeasured by scanning electron microscopy or atomic force microscopy,X-ray diffraction or Feigl solution analysis for positive staining orother known means for establishing the same, as will be appreciated bythe skilled artisan.

As described and exemplified herein, for example, as described inExamples 6, certain sections of isolated coral provide a differentphenotype, as compared to samples harvested from another region of alarger coral piece. Such phenotype may be reflected in the sample'sabsorptive capacity, surface structure roughness or both, with thestated difference resulting in a sample characterized in promoting celland tissue growth and/or restored function.

In some embodiments, the process for selection of the solid substratecomprises isolating a sample of a coralline-based solid material andestablishing the presence of a substantially rough surface on saidmarine organism skeletal derivative-based solid material, whichsubstantially rough surface is determined by scanning electronmicroscopy or atomic force microscopy, X-ray diffraction or Feiglsolution positive staining and selecting a marine organism skeletalderivative-based solid material characterized by a determination of thepresence of a substantially rough surface on said marine organismskeletal derivative-based solid material, or positive staining (black)via Feigl solution staining.

In some embodiments, coral-based solid substrates of this invention maybe converted to partially or fully into hydroxyapatite by known methods.

In another aspect, a solid substrate is converted to hydroxyapatite, andthe same is then assessed for the presence of substantial surfaceroughness (Ra) as measured by scanning electron microscopy or atomicforce microscopy or XRD analysis, and such substrates fulfilling thestated criteria are specifically selected and encompassed by the subjectapplication.

Without being bound by theory, and representing non-limiting embodimentsof the substrates, processes and applications of this invention,specific selection of marine organism skeletal derivative-based solidsubstrates characterized by the desired specific fluid uptake capacityvalue or desired surface roughness (Ra) as measured by methods fordetermining these characteristics as described herein, may select for asample whose vascularization is enhanced, or in some embodiments, whoseaccess to lymph is enhanced, or in some embodiments, whose absorptivecapacity heralds an affinity for extracellular matrix-associatedmaterials, or in some embodiments, whose absorptive capacity heralds anaffinity for cellular attraction including extravasation from proximalvessels.

In some embodiments, the specific selection of marine organism skeletalderivative-based solid substrates characterized by the desired specificfluid uptake capacity value or desired surface roughness (Ra) asmeasured by methods for determining these characteristics as describedherein, are useful in promoting anti-cancer activity. In someembodiments, the specific selection of marine organism skeletalderivative-based solid substrates characterized by the desired specificfluid uptake capacity value or desired surface roughness (Ra) asmeasured by methods for determining these characteristics as describedherein, are useful in promoting osteo-integration, osteo-conduction,osteo-transduction, chondrogenesis or cartilage regeneration, or acombination thereof. In some embodiments, the specific selection ofmarine organism skeletal derivative-based solid substrates characterizedby the desired specific fluid uptake capacity value or desired surfaceroughness (Ra) as measured by methods for determining thesecharacteristics as described herein, are useful in promoting ex-vivothree-dimensional structural support for cells, tissue or organ growth,which in some embodiments, is particularly suitable for applications inthe heart, muscle, liver, skin, kidney, blood vessel, or neurons.

It is to be understood that any of these mechanisms, and others, mayaccount for the phenomenon of enhanced cell or tissue growth or restoredfunction, and that any such mechanism associated with the application ofan optimized marine organism skeletal derivative-based solid substratefor promoting cell or tissue growth or restored function characterizedby the desired specific fluid uptake capacity value or desired surfaceroughness (Ra) as measured by methods for determining thesecharacteristics as described herein, is to be understood as being partof this invention.

In some embodiments, samples thus processed and found to becharacterized by the desired specific fluid uptake capacity value ordesired surface roughness (Ra) as measured by methods for determiningthese characteristics as described herein, may then be used for theisolation of proximally located regions of a section from which suchsample was taken, which samples can then be reliably used and consideredas being optimized in accordance with the processes of this invention.In some embodiments, with regard to coral-based samples, such regionsmay include the entire annual growth ring region within the coral fromwhich the sample was derived.

In some embodiments, samples thus processed and found to becharacterized by the desired specific fluid uptake capacity value ordesired surface roughness (Ra) as measured by methods for determiningthese characteristics as described herein, may then be utilized forimplantation into a subject or for use as an ex-vivo substrate for cellor tissue growth for subsequent implantation.

In some embodiments, the marine organism skeletal derivative-based solidsubstrates of this invention may be processed/prepared to form a bonefiller or bone substitute material.

In some embodiments, the marine organism skeletal derivative-based solidsubstrates of this invention are useful in orthopedic applications,including use as orthopedic screws, prostheses and others as will beappreciated by the skilled artisan. In some embodiments, the marineorganism skeletal derivative-based solid substrates of this inventionare useful in applications requiring a filler material, such as gapfiller.

In some embodiments, such fillers may include active glasses, as areknown to the skilled artisan. Other commercial products may be combinedwith the marine organism skeletal derivative-based solid substrates ofthis invention. In some embodiments, the bone filler materials describedin the following U.S. patents may be combined with the marine organismskeletal derivative-based solid substrates of this invention: U.S. Pat.Nos. 5,939,039; 6,325,987; 6,383,519; 6,521,246; 6,969,501; and6,991,803, all of which are hereby incorporated by reference in theirentirety.

In some embodiments, the marine organism skeletal derivative-based solidsubstrates of this invention are useful in applications making use ofreinforcing structures, for example as reinforcing screws, grafts, ofothers. In some embodiments, the methods and materials of this inventionare useful in fixation of screws, prosthesis and other structuressuitable for such application.

In some embodiments, the methods and materials of this invention areuseful in wound healing. In some embodiments, the materials of thisinvention include a solid marine organism skeletal derivative-basedsolid substrate, in accordance with any embodiment thereof as describedherein, including, inter alia, substrates on a millimeter or centimeterscale, or in some embodiments, use of powdered or granulated marineorganism skeletal derivative-based solid substrates are envisioned.

In some embodiments, such wound healing may include healing of burns,necrotic tissue, diabetic ulcers, surgical wounds, and any wound, aswill be appreciated by the skilled artisan.

In some embodiments, the methods and materials of this invention areuseful in applications in avascular necrosis, cyst or bone tumortreatment, for example, following surgical excision of same.

In some embodiments, the methods and materials of this invention areuseful in applications in cranio-facial skeletal surgery and skeletalreconstruction applications.

In other embodiments the substrate may be a mixture of several marineoriginated materials or a mixture of bone and coral granules orcartilage and coral granules. In some embodiments, the solid substratemay be a composite material comprised of multiple samples of the marineorganism skeletal derivatives as herein described.

In one embodiment of this invention, the solid substrate may be isolatedmarine organism skeletal derivative material alone, or in someembodiment, the substrate may further comprise an additional material.

In some embodiments, such additional material may include a polymer.

The term “polymer” refers, in some embodiments, to the presence of alayer of polymeric material in association with at least a portion ofthe solid substrate material. In some embodiments, such polymer layer isa coating for the solid substrate material.

In some embodiments, such coating may be over the entirety of the solidsubstrate, and in some embodiments, such coating may penetrate to withinthe voids and/or pores and/or hollows of the solid substrate. In someembodiments, such coating may be selectively applied to a particularregion of the solid substrate, such that it creates a separate phase onthe solid substrate, and in some embodiments, such polymer may be soapplied that a thick polymer layer or phase is associated with a portionof a solid substrate, thereby creating a separate polymer phase inassociation with the solid substrate as herein described.

In one embodiment, the polymer coating provides added features to thesolid substrates as herein described, for example, added tensilestrength, added flexibility, reduced brittleness, and other attributes,to the solid substrate and in some embodiments, the polymer coatingresults in greater cellular attraction and attachment to the solidsubstrates as herein described, which in turn, inter alia, results inenhanced repair in terms of quantity, quality and timing of repair. Insome embodiments, the polymer coating enhance cells proliferation and/ordifferentiation into desired mature tissue which in turn, inter alia,results in enhanced repair in terms of quantity, quality and timing ofrepair.

In one embodiment of this invention, a polymer coating is permeable. Inone embodiment, the permeable polymer coating comprises a special porousmembrane. In one embodiment, the term “permeable” refers to having poresand openings. In one embodiment, the permeable polymer coating of thisinvention has pores and openings which allow entry of nutrients, atherapeutic compound, a cell population, a chelator, or a combinationthereof. In one embodiment, the permeable polymer coating of thisinvention has pores and openings which allow exit/release of nutrients,a therapeutic compound, a cell population, a chelator, or a combinationthereof.

In one embodiment, a polymer coating of this invention is discontinuous.In one embodiment, a region or a plurality of sub-regions of the coralof this invention comprise an absence of polymer coating, allowingdirect contact between the coral and the environment.

In some embodiments, the marine organism skeletal derivative-based solidmaterial comprises a biocompatible polymer attached to an outer surfaceof the substrate. In some embodiments, the solid substrate incorporatesa biocompatible polymer therewithin, which is associated with thearagonite or calcite component, via any physical or chemicalassociation. In some embodiments, the polymer is a part of a hydrogel,which is incorporated in the solid substrates of this invention. In someembodiments, such hydrogel-containing solid substrates may thereafter belyophilized or desiccated, and may thereafter be reconstituted.

In some embodiments of the solid substrates of this invention, thepolymer may be applied to the solid substrate so as to form a separatephase, or in some embodiments, the polymer may be applied as a layeronto the solid substrate, or in some embodiments, the solid substratemay comprise both polymer as an internal or externally associated layerwith a separate phase attached thereto comprising the same or adifferent polymeric material.

Such polymer-containing solid substrates may be particularly suited forcartilage repair, regeneration or enhancement of formation thereof. Insome embodiments, according to this aspect, for example, in thetreatment of osteochondral defects, the coralline-based solid substrateis of a dimension suitable for incorporation within affected bone, andfurther comprises a polymer-containing phase, which phase, when insertedwithin the affected defect site, is proximal to affected cartilage. Inanother aspect and representing an embodiment of this invention, thesolid substrate comprises a polymer, which has permeated within thevoids and pores of the solid substrate, which solid substrate isinserted within a site of cartilage repair and which polymer facilitatescartilage growth, regeneration or healing of the defect site.

Such polymer-containing solid substrates may be particularly suited forbone repair, regeneration or enhancement of formation thereof. In someembodiments, according to this aspect, for example, in the treatment ofbone edema, bone breakage or fragmentation, disease or defect, thecoralline-based solid substrate is of a dimension suitable forincorporation within affected bone, and further comprises a polymer,which polymer has permeated within the voids and pores of the solidsubstrate, which solid substrate is inserted within the bone and whichpolymer facilitates bone growth, regeneration or healing of the defectsite.

In one embodiment, the biocompatible polymer comprises a natural polymercomprising a glycosaminoglycan, collagen, fibrin, elastin, silk,chitosan, alginate, and any combination thereof. In one embodiment, apolymer coating of this invention comprises a natural polymercomprising, collagen, fibrin, elastin, silk, hyaluronic acid, sodiumhyaluronate, cross linked hyaluronic acid, chitosan, cross linkedchitosan, alginate, calcium alginate, cross linked calcium alginate andany combination thereof.

In one embodiment, the polymer comprises synthetically modified naturalpolymers, and may include cellulose derivatives such as alkylcelluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose estersand nitrocelluloses. Examples of suitable cellulose derivatives includemethyl cellulose, ethyl cellulose, hydroxypropyl cellulose,hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, celluloseacetate, cellulose propionate, cellulose acetate butyrate, celluloseacetate phthalate, carboxymethyl cellulose, cellulose triacetate andcellulose sulfate sodium salt.

In one embodiment, of this invention, a polymer comprises a syntheticbiodegradable polymer. In one embodiment of this invention, a syntheticbiodegradable polymer comprises alpha-hydroxy acids includingpoly-lactic acid, polyglycolic acid, enantiomers thereof, co-polymersthereof, polyorthoesters, and combinations thereof.

In one embodiment, a polymer of this invention comprises apoly(cianoacrylate), poly(alkyl-cianoacrylate), poly(ketal),poly(caprolactone), poly(acetal), poly(α-hydroxy-ester),poly(α-hydroxy-ester), poly(hydroxyl-alkanoate),poly(propylene-fumarate), poly (imino-carbonate), poly(ester),poly(ethers), poly(carbonates), poly(amide), poly(siloxane),poly(silane), poly(sulfide), poly(imides), poly(urea),poly(amide-enamine), poly(organic acid), poly(electrolytes),poly(p-dioxanone), poly(olefin), poloxamer, inorganic or organomatallicpolymers, elastomer, or any of their derivatives, or a copolymerobtained by a combination thereof.

In one embodiment, a polymer of this invention comprisespoly(D,L-lactide-co-glycolide) (PLGA). In another embodiment, thepolymer comprises poly(D,L-lactide) (PLA). In another embodiment, thepolymer comprises poly(D,L-glycolide) (PGA). In one embodiment, thepolymer comprises a glycosaminoglycan.

In one embodiment, the polymer comprises synthetic degradable polymers,which may include, but are not limited to polyhydroxy acids, such aspoly(lactide)s, poly(glycolide)s and copolymers thereof; poly(ethyleneterephthalate); poly(hydroxybutyric acid); poly(hydroxyvaleric acid);poly[lactide-co-(ε-caprolactone)]; poly[glycolide-co(ε-caprolactone)];poly(carbonate)s, poly(pseudo amino acids); poly(amino acids);poly(hydroxyalkanoate)s; poly(anhydrides); poly(ortho ester)s; andblends and copolymers thereof.

In one embodiment of this invention, a polymer comprises proteins suchas zein, modified zein, casein, gelatin, gluten, serum albumin,collagen, actin, α-fetoprotein, globulin, macroglobulin, cohesin,laminin, fibronectin, fibrinogen, osteocalcin, osteopontin,osteoprotegerin, or others, as will be appreciated by one skilled in theart. In another embodiment, a polymer may comprise cyclic sugars,cyclodextrins, synthetic derivatives of cyclodextrins, glycolipids,glycosaminoglycans, oligosaccharide, polysaccharides such as alginate,carrageenan (χ, λ, μ, κ), chitosan, celluloses, chondroitin sulfate,curdlan, dextrans, elsinan, furcellaran, galactomannan, gellan,glycogen, arabic gum, hemicellulose, inulin, karaya gum, levan, pectin,pollulan, pullulane, prophyran, scleroglucan, starch, tragacanth gum,welan, xanthan, xylan, xyloglucan, hyaluronic acid, chitin, or apoly(3-hydroxyalkanoate)s, such as poly(β-hydroxybutyrate),poly(3-hydroxyoctanoate) or poly(3-hydroxyfatty acids), or anycombination thereof.

In one embodiment, the polymer comprises a bioerodible polymer such aspoly(lactide-co-glycolide)s, poly(anhydride)s, and poly(orthoester)s,which have carboxylic groups exposed on the external surface as thesmooth surface of the polymer erodes, which may also be used. In oneembodiment, the polymer contains labile bonds, such as polyanhydridesand polyesters.

In one embodiment, a polymer may comprise chemical derivatives thereof(substitutions, additions, and elimination of chemical groups, forexample, alkyl, alkylene, hydroxylations, oxidations, and othermodifications routinely made by those skilled in the art), blends of,e.g. proteins or carbohydrates alone or in combination with syntheticpolymers.

In one embodiment of this invention, the polymer is biodegradable. Inone embodiment, the term “biodegradable” or grammatical forms thereof,refers to a material of this invention, which is degraded in thebiological environment of the subject in which it is found. In oneembodiment, the biodegradable material undergoes degradation, duringwhich, acidic products, or in another embodiment, basic products arereleased. In one embodiment, bio-degradation involves the degradation ofa material into its component subunits, via, for example, digestion, bya biochemical process. In one embodiment, biodegradation may involvecleavage of bonds (whether covalent or otherwise), for example in apolymer backbone of this invention. In another embodiment,biodegradation may involve cleavage of a bond (whether covalent orotherwise) internal to a side-chain or one that connects a side chainto, for example a polymer backbone.

In one embodiment, a coral of this invention is covalently associatedwith the polymer coating via the use of a cross-linking agent. In oneembodiment, the phrase “cross-linking agent” refers to an agent whichfacilitates the formation of a covalent bond between 2 atoms. In oneembodiment, the cross-linking agent is a zero-length cross-linkingagent.

In one embodiment, the cross-linking agent is (1 ethyl 3-(3dimethylaminopropyl)carbodiimide (EDAC), N-Sulfohydroxy succinamide (Sulfo NHS),5-iodopyrimidines, N-carbalkoxydihydroquinolines,pyrroloquinolinequinones, genipin or a combination thereof.

In one embodiment, the cross-linking agent is a homobifunctionalcross-linker, such as, for example, a N-hydroxysuccinimide ester (e.g.disuccinimidyl suberate or dithiobis(succinimidylpropionate),homobifunctional imidoester (e.g. dimethyladipimidate or dimethylpimelimidate), sulfhydryl-reactive crosslinker (e.g.1,4-di-[3′-(2′-pyridyldithio)propionamido]butane), difluorobenzenederivative (e.g. 1,5-difluoro-2,4-dinitrobenzene), aldehyde (e.g.formaldehyde, glutaraldehyde), bis-epoxide (e.g. 1,4-butanedioldiglycidyl ether), hydrazide (e.g. adipic acid dihydrazide),bis-diazonium derivative (e.g. o-tolidine), bis-alkylhalide, or acombination thereof.

In one embodiment, the cross-linking agent is a heterobifunctionalcross-linker, such as, for example, an amine-reactive andsulfhydryl-reactive crosslinker (e.g. N-succinimidyl3-(2-pyridyldithio)propionate, a carbonyl-reactive andsulfhydryl-reactive crosslinker (e.g. 4-(4-N-maleimidophenyl)butyricacid hydrazide), or a combination thereof.

In some embodiments, the cross-linking agent is a trifunctionalcross-linkers, such as, for example,4-azido-2-nitrophenylbiocytin-4-nitrophenyl ester,sulfosuccinimidyl-2-[6-biotinamido]-2-(p-azidobenzamido)hexanoamido]ethyl-1,3′-dithiopropionate(sulfo-SBED), or a combination thereof.

In another embodiment, the cross-linking agent is an enzyme. In oneembodiment of this invention, the cross-linking agent comprises atransglutaminase, a peroxidase, a xanthine oxidase, a polymerase, or aligase, or a combination thereof.

The choice of concentration of the cross-linking agent utilized foractivity will vary, as a function of the volume, agent and polymerchosen, in a given application, as will be appreciated by one skilled inthe art.

In one embodiment, the association of a coral of this invention with apolymer coating of this invention comprises a physical and/or mechanicalassociation. For example, in one embodiment, a physical and/ormechanical association may comprise imbibing of any means, air drying,using a cross-linking agent, applying of heat, applying vacuum, applyinglyophilizing methods, freezing, applying mechanical forces or anycombination thereof, to promote the physical association between a coraland a polymer coating as described herein.

In some embodiments, the choice of polymer, or application of polymer toa solid substrate as herein described may be so chosen, for an addedability to increase fluid uptake. Similarly, the surface of the solidsubstrate may be treated to increase fluid uptake therewithin, as well.In some embodiments, such surface treatment may include application ofplasma to the solid substrate.

It will be apparent to one skilled in the art that the physical and/orchemical properties of a polymer application to a solid substrate ofthis invention and components thereof may influence methods of use ofthis invention and kits thereof, for inducing or enhancing cartilageand/or bone repair.

In one embodiment, the polymer as applied to the solid substrates ofthis invention has a thickness of between 2.0 μm and 0.1 μm. In oneembodiment, the polymer coating has a thickness of about 1.0 μm. In oneembodiment, the polymer coating of this invention has a thickness ofbetween 10 μm and 50 μm. In one embodiment, the polymer coating asapplied to the solid substrates of this invention has a thickness ofabout 10-25, or about 15-30, or about 25-50 μm. In one embodiment, thepolymer coating as applied to the solid substrates of this invention hasa thickness of about 0.0001-0.1 μm. In one embodiment, the polymercoating as applied to the solid substrates of this invention has athickness of about 20-200 μm. In one embodiment, the polymer coating asapplied to the solid substrates of this invention has a thickness ofabout 100-1500 μm.

In some embodiments, the polymer as applied to the solid substrates ofthis invention is a thin coating, which is associated with the solidsubstrates of this invention and has a thickness as indicatedhereinabove.

In some embodiments, the polymer as applied to the solid substrates ofthis invention is applied throughout the solid substrates of thisinvention, such that, in some embodiments, the pores and voids withinthe solid substrates of the invention may be filled with polymers asherein described, and such polymer layer as applied may have a thicknessof about 60-900 μm.

In some embodiments, the polymer as applied to the solid substrates ofthis invention is to a terminus or a portion of the coating forming anadditional polymer phase on the solid substrates of the invention.According to this aspect, and in some embodiments, the polymer layer asapplied will have a thickness of between about 0.01-10 mm.

In some embodiments, multiple solid substrates comprising polymericadditives are implanted into a desired implantation site, wherein thepolymer thickness applied to a first solid substrate may vary ascompared to a polymer thickness as applied to a second solid substrate,implanted in the desired site. Variations in such thickness may reflectthe range described herein.

In one embodiment, the thickness of the polymer as applied to the solidsubstrates of this invention influences physical characteristics of asolid substrate of this invention. For example, the thickness of apolymeric application may influence elasticity, tensile strength,adhesiveness, or retentiveness, or any combination thereof of a solidsubstrate of this invention. In one embodiment, the polymer applicationincreases the elasticity of a solid substrate of this invention. In oneembodiment, a polymeric application increases the tensile strength of asolid substrate of this invention. In one embodiment, the adhesivenessof a polymeric application relates to adhesion of mesenchymal stemcells, blood vessels, tissue at a site of desired repair, includingcartilage repair, cartilage tissue, or bone tissue, or a combinationthereof. In one embodiment, a polymeric application decreases theadhesiveness of a solid substrate of this invention. In one embodiment,a polymeric application increases the adhesiveness of a solid substrateof this invention. One skilled in the art will recognize that apolymeric application may increase adhesiveness for an item whiledecreasing adhesiveness for another item. For example, in oneembodiment, the polymeric application increases adhesiveness for amesenchymal stem cell and decreases adhesiveness of an infective agent.In one embodiment, the retentiveness of a polymeric application relatesto retention of a cell population. In one embodiment, the cellpopulation retained within a polymer coating is a mesenchymal stem cellpopulation, chondrocyte population osteoblast population, etc. In oneembodiment, the retentiveness of a polymeric application relates toretention of effector compounds.

In one embodiment, the thickness of the polymeric application influencesproliferation and/or differentiation of cells applied to the solidsubstrates of this invention, or influences the activation or migrationof cells associated with cell or tissue growth/restored function to thesubstrates of this invention, or a combination thereof.

Incorporation of a biocompatible polymer such as hyaluronic acid withina solid substrate of this invention may be accomplished via any means,including, in some embodiments, pressure-driven application, forexample, via application under vacuum, centrifugal force or mechanicalpressure. In some embodiments, gravitational force is sufficient toallow appropriate and relatively homogenous penetration of thehyaluronic acid to a desired depth of the implant. According to thisaspect, in one embodiment, visual inspection of the implant, for exampleusing the staining with Fast Green/Safranin O, demonstrates uniformdistribution of the hyaluronic acid through the substrate to a desireddepth as a function of the time and conditions of application.

In one embodiment, the solid substrates of this invention may furthercomprise an effector compound, which in some embodiments, may beassociated directly with the solid substrates of this invention, or insome embodiments, may be associated with a polymer, and applied inconnection therewith.

In one embodiment, such effector compounds might include silver ions,copper ions or other metals, or combinations thereof. In anotherembodiment release of this compound might be facilitated by theapplication of electrical charge.

In another embodiment a first implant may be coated with a metal such assilver and a second implant may be coated with a second metal such asgold. Application of electrical field or actuation by battery mightcause an electrical charge to flow between the implanted materials andlead to sterilization of the area due to discharge of silver ions. Suchimplementation might, for example, be useful in the treatment ofosteomyelitis.

In one embodiment, the effector compound comprises a component of a kitof this invention for use for incorporation into a solid substrate ofthis invention as herein described.

In one embodiment of this invention, the effector compound comprises acytokine, a bone morphogenetic protein (BMP), growth factors, achelator, a cell population, a therapeutic compound, or an antibiotic,or any combination thereof.

In one embodiment of this invention, the phrase “a therapeutic compound”refers to a peptide, a protein or a nucleic acid, or a combinationthereof. In another embodiment, the therapeutic compound is anantibacterial, antiviral, antifungal or antiparasitic compound. Inanother embodiment, the therapeutic compound has cytotoxic oranti-cancer activity. In another embodiment, the therapeutic compound isan enzyme, a receptor, a channel protein, a hormone, a cytokine or agrowth factor. In another embodiment, the therapeutic compound isimmune-stimulatory. In another embodiment, the therapeutic compoundinhibits inflammatory or immune responses. In one embodiment, thetherapeutic compound comprises a pro-angiogenic factor. In oneembodiment, the therapeutic compound or drug comprises ananti-inflammatory compound, an anti-infective compound, a pro-angiogenicfactors or a combination thereof.

In one embodiment, the effector compound comprises, an anti-helminth, anantihistamine, an immune-modulatory, an anticoagulant, a surfactant, anantibody, a beta-adrenergic receptor inhibitor, a calcium channelblocker, an ace inhibitor, a growth factor, a hormone, a DNA, an siRNA,or a vector or any combination thereof.

In one embodiment, the phrase “effector compound” refers to any agent orcompound, which has a specific purpose or application which is useful inthe treatment, prevention, inhibition, suppression, delay or reductionof incidence of infection, a disease, a disorder, or a condition, whenapplied to the solid substrates, kits and/or methods of this invention.An effector compound of this invention, in one embodiment, will producea desired effect which is exclusive to the ability to image thecompound. In some embodiments, the effector compound may be useful inimaging a site at which the compound is present, however, such abilityis secondary to the purpose or choice of use of the compound.

In one embodiment of this invention, term “effector compound” is to beunderstood to include the terms “drug” and “agent”, as well, whenreferred to herein, and represents a molecule whose incorporation withinthe solid substrate and/or kits of this invention, or whose use thereof,is desired. In one embodiment, the agent is incorporated directly withina solid substrate, and/or kit of this invention. In another embodiment,the agent is incorporated within a solid substrate and/or kit of thisinvention, either by physical interaction with a polymer coating, acoral, or coral particles of this invention, and/or a kit of thisinvention, or association thereto.

In one embodiment, the “effector compound” is a therapeutic compound.

In one embodiment, the phrase “a therapeutic compound”, refers to amolecule, which when provided to a subject in need, provides abeneficial effect. In some cases, the molecule is therapeutic in that itfunctions to replace an absence or diminished presence of such amolecule in a subject. In one embodiment, the molecule is a nucleic acidcoding for the expression of a protein is absent, such as in cases of anendogenous null mutant being compensated for by expression of theforeign protein. In other embodiments, the endogenous protein ismutated, and produces a non-functional protein, compensated for by theexpression of a heterologous functional protein. In other embodiments,expression of a heterologous protein is additive to low endogenouslevels, resulting in cumulative enhanced expression of a given protein.In other embodiments, the molecule stimulates a signaling cascade thatprovides for expression, or secretion, or others of a critical elementfor cellular or host functioning.

In another embodiment, the therapeutic compound may be natural ornon-natural insulins, amylases, proteases, lipases, kinases,phosphatases, glycosyl transferases, trypsinogen, chymotrypsinogen,carboxypeptidases, hormones, ribonucleases, deoxyribonucleases,triacylglycerol lipase, phospholipase A2, elastases, amylases, bloodclotting factors, UDP glucuronyl transferases, ornithinetranscarbamoylases, cytochrome p450 enzymes, adenosine deaminases, serumthymic factors, thymic humoral factors, thymopoietins, growth hormones,somatomedins, costimulatory factors, antibodies, colony stimulatingfactors, erythropoietin, epidermal growth factors, hepaticerythropoietic factors (hepatopoietin), liver-cell growth factors,interleukins, interferons, negative growth factors, fibroblast growthfactors, transforming growth factors of the α family, transforminggrowth factors of the β family, gastrins, secretins, cholecystokinins,somatostatins, serotonins, substance P, transcription factors orcombinations thereof.

In any of the embodiments herein, coralline solid substrates, and theiruse in the methods of the present invention may further comprise, or beimplanted with, other compounds such as, for example, antioxidants,growth factors, cytokines, antibiotics, anti-inflammatories,immunosuppressors, preservative, pain medication, other therapeutics,and excipient agents. In one embodiment, examples of growth factors thatmay be administered in addition to the HMG-CoA reductase inhibitorinclude, but are not limited to, epidermal growth factor (EGF),transforming growth factor-alpha (TGF-α), transforming growthfactor-beta (TGF-β), human endothelial cell growth factor (ECGF),granulocyte macrophage colony stimulating factor (GM-CSF), bonemorphogenetic protein (BMP), nerve growth factor (NGF), vascularendothelial growth factor (VEGF), fibroblast growth factor (FGF),insulin-like growth factor (IGF), cartilage derived morphogeneticprotein (CDMP), platelet rich plasma (PRP), platelet derived growthfactor (PDGF), or any combinations thereof. Examples of antibioticsinclude antimicrobials and antibacterials.

In one embodiment, effector compounds for use in a solid substrateand/or a kit of this invention and/or a method of this invention maycomprise, inter-alia, an antibody or antibody fragment, a peptide, anoligonucleotide, a ligand for a biological target, an immunoconjugate, achemomimetic functional group, a glycolipid, a labelling agent, anenzyme, a metal ion chelate, an enzyme cofactor, a cytotoxic compound, abactericidal compound, a bacteriostatic compound, a fungicidal compound,a fungistatic compound, a chemotherapeutic, a growth factor, a hormone,a cytokine, a toxin, a prodrug, an antimetabolite, a microtubuleinhibitor, a radioactive material, or a targeting moiety, or anycombination thereof.

In one embodiment, the solid substrates and/or kits of this inventionand/or methods of this invention comprise or make use of anoligonucleotide, a nucleic acid, or a vector. In some embodiments, theterm “oligonucleotide” is interchangeable with the term “nucleic acid”,and may refer to a molecule, which may include, but is not limited to,prokaryotic sequences, eukaryotic mRNA, cDNA from eukaryotic mRNA,genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and evensynthetic DNA sequences. The term also refers to sequences that includeany of the known base analogs of DNA and RNA.

The solid substrates and/or kits of this invention and/or methods of useof this invention may comprise nucleic acids, in one embodiment, or inanother embodiment, the solid substrates and/or kits of this inventionand/or methods of use of this invention may include delivery of thesame, as a part of a particular vector. In one embodiment,polynucleotide segments encoding sequences of interest can be ligatedinto commercially available expression vector systems suitable fortransducing/transforming mammalian cells and for directing theexpression of recombinant products within the transduced cells. It willbe appreciated that such commercially available vector systems caneasily be modified via commonly used recombinant techniques in order toreplace, duplicate or mutate existing promoter or enhancer sequencesand/or introduce any additional polynucleotide sequences such as forexample, sequences encoding additional selection markers or sequencesencoding reporter polypeptides.

In one embodiment, effector compounds for use in a solid substrateand/or a kit of this invention and/or a method of this invention maycomprise, inter-alia, a cytokine, a bone morphogenetic protein (BMP),growth factor, a chelator, a cell population, a therapeutic compound, ananti-inflammatory compound, a pro-angiogenic compound or an antibiotic,or any combination thereof.

In some embodiments, the kits and/or marine organism skeletalderivative-based solid substrates of this invention comprise knownosteoinductive materials, bone cements, bone glasses, or bone fillers ora combination thereof.

In some embodiments, the bone cements may include any known cement,including β-Tricalcium phosphate, Monocalcium phosphate monohydrate(MCPM) (Ca(H2PO4)2H2O) and mixtures thereof, including Brucite cement.In some embodiments, the cement may include amorphous calcium phosphate(ACP), dicalcium phosphate dihydrate (DCPD), dicalcium phosphateanhydrous (DCPA), α-tricalcium phosphate (α-TCP), dicalcium phosphate(DCP), tetracalcium phosphate (TTCP), monocalcium phosphate monohydrate(MCPM), calcium carbonate (CC) and others, and mixtures thereof.

In some embodiments, the kits and/or marine organism skeletalderivative-based solid substrates of this invention comprise known, andin some embodiments, commercially available osteoinductive materials,including, for example, bioactive glasses, bone cement components suchas β-TCP, poly(-methyl methacrylate).

In some embodiments, the solid substrates of this invention may beseeded with cells, cell populations or tissue.

In some embodiments, the cells or tissue comprise stem or progenitorcells, or a combination thereof.

In one embodiment of this invention, the cells or tissue as used inaccordance with the substrates, methods of use or kits of thisinvention, are engineered to express a desired product.

In one embodiment, the phrase “a cell population” refers to atransfected cell population, a transduced cell population, a transformedcell population, or a cell population isolated from a subject, or acombination thereof. In some embodiments, transfected, transduced ortransformed cells, may be seeded on the solid substrate, or in someembodiments, may be incorporated into a polymeric application thereto,or a combination thereof.

In one embodiment, a cell population of this invention comprisesmesenchymal stem cells. In one embodiment, the mesenchymal stem cellsare transformed.

In one embodiment, a cell population comprises cells beneficial inrepair of a tissue for which the implantation of a solid substrate ofthis invention is desired.

In some embodiments, the cells are beneficial in and/or promotecartilage and/or bone formation and/or repair. Such cells may includechondroblasts or chondrocytes; fibrochondrocyte; osteocyte; osteoblast;osteoclast; synoviocyte; bone marrow cell; stromal cell; stem cell;embryonic stem cell; precursor cell, derived from adipose tissue;peripheral blood progenitor cell; stem cell isolated from adult tissue;genetically transformed cell; or a combination thereof. In anotherembodiment, a precursor cell may refer to a combination of chondrocytesand other cells; a combination of osteocytes and other cells; acombination of synoviocytes and other cells; a combination of bonemarrow cells and other cells; a combination of mesenchymal cells andother cells; a combination of stromal cells and other cells; acombination of stem cells and other cells; a combination of embryonicstem cells and other cells; a combination of precursor cells isolatedfrom adult tissue and other cells; a combination of peripheral bloodprogenitor cells and other cells; a combination of stem cells isolatedfrom adult tissue and other cells; and a combination of geneticallytransformed cells and other cells. the precursor cells for use in themethod of the present invention are prepared from an organ tissue of therecipient mammal (i.e. autologous), or a syngeneic mammal. In anotherembodiment, allogeneic and xenogeneic precursor cells may be utilized.

In one embodiment, the solid substrate of this invention incorporatesstem or progenitor or precursor cells. Such cells can be obtaineddirectly from a mammalian donor, e.g., a patient's own cells, from aculture of cells from a donor, or from established cell culture lines.In some embodiments, the mammal is a mouse, rat, rabbit, guinea pig,hamster, cow, pig, horse, goat, sheep, dog, cat, monkey, ape or a human.Cells of the same species and/or of the same immunological profile canbe obtained by biopsy, either from the patient or a close relative.Using standard cell culture techniques and conditions, the cells arethen grown in culture until confluent and used when needed. The cellsmay be cultured until a sufficient number of cells have been obtainedfor a particular application.

In one embodiment, the solid substrate of this invention incorporatesany cell which may participate in tissue repair, for example, incartilage and/or bone formation or repair. In some embodiments, suchcells represent autografts, in that cells are cultured ex-vivo to seedthe cells on the solid substrates of the invention, and such seededsolid substrates are implanted into the subject.

In some embodiments, such cells may represent allografts or xenografts,which may be incorporated within the solid substrates of this inventionand implanted within a site of repair.

In one embodiment, a coral of this invention comprises a cell populationfrom in vitro culture of the coral for a time period sufficient to seedthe cells in the coral. In one embodiment, the cell population is amesenchymal stem cell population, chondrocyte; fibrochondrocyte;osteocyte; osteoblast; osteoclast; synoviocyte; bone marrow cell;stromal cell; stem cell; embryonic stem cell; precursor cell, derivedfrom adipose tissue; peripheral blood progenitor cell; stem cellisolated from adult tissue; genetically transformed cell; or acombination thereof. In one embodiment, the mesenchymal stem cells;chondrocyte; fibrochondrocyte; osteocyte; osteoblast; osteoclast;synoviocyte; bone marrow cell; stromal cell; stem cell; embryonic stemcell; precursor cell, derived from adipose tissue; peripheral bloodprogenitor cell; stem cell isolated from adult tissue; geneticallytransformed cell; or a combination thereof seeded in vitro aretransformed. In one embodiment, the cell population comprises a cellpopulation beneficial for cartilage repair. In one embodiment, theculture comprises a chelator. In one embodiment of this invention, thechelator in a culture comprises a calcium chelator.

In some embodiments, the solid substrate may further serve as a bonesubstitute or bone void filler. In some embodiments, the solid substratemay further incorporate a bone-substitute or bone void filler. In someembodiments, such bone-containing material may comprise autologous orallogeneic bone. In some embodiments, such bone-containing material maycomprise animal bone.

As exemplified herein, blood, water and other hydrophilic fluids asdescribed were applied to the coral samples and absorption of the fluidwithin the coral samples was assessed.

FIGS. 1A-1F depict the results of a representative absorption studiesconducted as described, showing patterns of uptake, substantial uptakeand partial, minimal or no uptake of a fluid, respectively, dependingupon the sample assessed. This variability in the pattern of absorptionsurprisingly provided a means of selecting solid substrates withoptimized efficacy in cell and tissue growth and/or restored functionfollowing implantation.

Example 2 as provided herein demonstrates correlation betweensubstantial uptake of biological fluid within the implanted coral solidsubstrate and subsequent healing at the implantation site, in asurprisingly superior manner, as compared to coral solid substrates,which had minimal biological fluid uptake.

Also exemplified is the development of a screening protocol establishedto select for such optimized coral-based solid substrate for promotingcell or tissue growth, provided in Example 3.

Example 5 provides support for the characterization of organism skeletalderivative-based solid substrates having a contact angle value of lessthan 60 degrees, when in contact with a fluid, as being a comparableselection means as that provided by the specific fluid uptake capacityvalue, as described hereinabove.

This invention provides the unexpected application of optimally selectedcoral based solid substrates being useful in cell and tissue growthand/or restored function, and exemplified herein is the particularapplication for cartilage and bone repair and enhancement of formation.

In particular, this invention provides the unexpected application thatbone regeneration, (optionally with osteo-integration, osteo-conductionand osteo-transduction) repair and enhancement of formation is optimalwhen the solid substrate is characterized by a specific fluid uptakecapacity value of at least 75%, which specific fluid uptake capacityvalue was determined by establishing a spontaneous fluid uptake valuedivided by a total fluid uptake value and such substrate is insertedwithin a site for bone repair.

In particular, this invention provides the unexpected application thatbone regeneration, repair and enhancement of formation is optimal whenthe solid substrate is characterized by having a contact angle value ofless than 60 degrees, when in contact with a fluid and such substrate isinserted within a site for bone repair.

In other embodiments, this invention provides the unexpected advantagein terms of greater chondrogenesis, when the solid substrate ischaracterized by a specific fluid uptake capacity value of at least 75%,which specific fluid uptake capacity value was determined byestablishing a spontaneous fluid uptake value divided by a total fluiduptake value and such substrate was inserted within the cartilage defectsite.

In other embodiments, this invention provides the unexpected advantagein terms of greater chondrogenesis, when the solid substrate ischaracterized by having a contact angle value of less than 60 degrees,when in contact with a fluid and such substrate was inserted within thecartilage defect site.

This invention in some embodiments also provides the unexpectedapplication of optimally selected coral based solid substrates beinguseful in cell and tissue growth and/or restored function, andexemplified herein is the particular application for cartilage and bonerepair and enhancement of formation.

In particular, this invention provides the unexpected application thatbone regeneration, repair and enhancement of formation optionally withosteointegration, osteoconduction and osteotransduction is optimal whenthe solid substrate is characterized by a substantial surface roughness(Ra) as measured by scanning electron microscopy or atomic forcemicroscopy or XRD analysis and such substrate is inserted within a sitefor bone repair.

In other embodiments, this invention provides the unexpected advantagein terms of greater chondrogenesis, when the solid substrate ischaracterized by substantial surface roughness (Ra) as measured byscanning electron microscopy or atomic force microscopy or XRD analysisand such substrate was inserted within the cartilage defect site.

In some embodiments, solid substrates of this invention may be appliedfor use in a subject with a bone defect in need of repair, whereinaccess to the bone defect results in the creation of a defect in theoverlying cartilage, and the solid substrates of this invention allowfor the healing of affected bone or bone and cartilage tissues.

In other embodiments, such solid substrates may be administered to asubject with a cartilage defect in need of repair, wherein optimalinsertion of the solid substrate for stimulation of cartilage repairnecessitates anchoring of the scaffold in the underlying bone, forexample, by creating void in the underlying bone for insertion of thesolid substrates, and once inserted, the solid substrate facilitatesrepair of both the overlying cartilage and underlying bone.

In other embodiments, such solid substrate may be administered to asubject with an osteochondral defect, where both bone and cartilagetissue are in need of repair as part of the pathogenesis of thedisorder. The solid substrates according to this aspect are in someembodiments, particularly suited for such applications.

It will be appreciated by the skilled artisan, that the applications, inparticular, as related to bone therapy may include use of a solidsubstrate that incorporates any additional element as described herein,including, for example, bone allograft, bone autograft, bonesubstitutes, known bone void fillers, therapeutic compounds, and thelike.

In some embodiments, the solid substrates of this invention may be usedin conjunction with other known and/or available materials forstimulating/enhancing cell and/or tissue growth and/or restoredfunction, for example, by promoting bone and/or cartilage repair.

In some embodiments, the solid substrates of this invention may beutilized to affix additional solid substrates, for example for use inwhole joint repair or ligament repair, or other connector tissue repair.

In some embodiments, the solid substrates of this invention may be usedfor example, as a pin, in conjunction with other scaffolds for bonerepair or regeneration, etc. It is to be understood that any use of thesolid substrates of this invention, alone or in conjunction with otherappropriate materials, for the treatment, repair or stimulation of cellor tissue growth or restored function is to be considered as part ofthis invention

It will be appreciated that the solid substrates of this invention maybe of any suitable shape or size to accommodate its application inaccordance with the methods of this invention. For example, and in someembodiments, for applications of the solid substrates of this inventionwithin long bones of a subject, the dimensions of the solid substratewill be scaled to approximate that of the site into which the scaffoldwill be implanted, and may be on an order of magnitude scaling frommillimeters to centimeters, as needed. Similarly, shapes of the solidsubstrates of the invention may be any shape into which the solidsubstrates of this invention may be machined or processed, and may haveany configuration as will be appropriate to achieve the desiredapplication for cell and/or tissue growth and restored function.

In one embodiment, the coral is shaped in the form of the tissue to begrown. For example, the coral can be shaped as a piece of cartilaginoustissue, such as a meniscus for a knee or elbow; vertebra, spineapplications, skull, disk, a joint; an articular surface of a bone, therib cage, a hip, a pelvis, an ear, a nose, a ligament, the bronchialtubes and the intervertebral discs.

This invention provides, in some embodiments, coralline solid substratesfor use in repairing cartilage and/or bone tissue defects associatedwith physical trauma or cartilage and/or bone tissue defects associatedwith a disease or disorder in a subject.

In one embodiment, the coralline solid substrate is shaped prior to usein a method of cartilage and/or bone repair. In one embodiment, thecoralline solid substrates is shaped concurrent with a method ofcartilage and/or bone repair, e.g., the coralline solid substrates maybe shaped during surgery when the site of repair may be best observed,thus optimizing the shape of the coralline solid substrates used.

In some embodiments, multiple coralline solid substrates are inserted tomaximally occupy a defect site, such that each coralline solid substratemay be inserted at a different angle and/or shape and/or depth and/orporosity to accommodate proper insertion into the desired region withina desired implantation site. It is to be understood that the referenceto angles or positioning may be with regard to one or more corallinesolid substrates inserted in a particular implantation site.

In one embodiment, the phrase “cartilage repair” refers to restoring acartilage defect to a more healthful state. In one embodiment, restoringcartilage results in regeneration of cartilage tissue. In oneembodiment, restoring cartilage results in regeneration of a full orpartial thickness articular cartilage defect. In one embodiment,restoring cartilage results in complete or partial regeneration ofcartilage tissue at a site of cartilage repair. In one embodiment,cartilage repair may result in restoration/repair of missing ordefective bone tissue, wherein repair of a cartilage defect necessitatesremoval of bone tissue at a site of cartilage repair. In one embodiment,restoring cartilage results in regeneration of osteochondral defect. Inone embodiment, cartilage repair comprises restoring cartilage defectsof joints (e.g. knee, elbow, hip, shoulder joints), of ears, of a nose,or of a wind pipe.

In some embodiments, the “cartilage repair” refers to treating,preventing or ameliorating or abrogating the symptoms of, orameliorating or abrogating the pathogenesis of osteoarthritis anddegenerative changes in cartilage.

In one embodiment, the phrase “bone repair” refers to restoring a bonedefect to a more healthful state. In one embodiment, restoring boneresults in regeneration of bone tissue. In one embodiment, restoringbone results in the filling in of any fracture or void within a bonetissue. In one embodiment, restoring bone results in complete or partialregeneration of bone tissue at a site of bone repair. In one embodiment,bone repair may result in restoration/repair of missing or defectivebone tissue. In one embodiment, bone repair comprises restoring bonedefects of any bone, treating bone edema, and other bone disorders, asneeded.

In some embodiments, the phrase “bone repair” refers to the treatment ofa subject with osteoporosis, Paget's disease, fibrous dysplasias, boneedema or osteodystrophies. In another embodiment, the subject has boneand/or cartilage infirmity. In another embodiment, the subject has otherbone remodeling disorders include osteomalacia, rickets, rheumatoidarthritis, achondroplasia, osteochodrytis, hyperparathyroidism,osteogenesis imperfecta, congenital hypophosphatasia, fribromatouslesions, multiple myeloma, abnormal bone turnover, osteolytic bonedisease, periodontal disease, or a combination thereof. In oneembodiment, bone remodeling disorders include metabolic bone diseaseswhich are characterized by disturbances in the organic matrix, bonemineralization, bone remodeling, endocrine, nutritional and otherfactors which regulate skeletal and mineral homeostasis, or acombination thereof. Such disorders may be hereditary or acquired and inone embodiment, are systemic and affect the entire skeletal system.

In some embodiments, the phrase “bone repair” refers to treating,preventing or ameliorating or abrogating the symptoms of, orameliorating or abrogating the pathogenesis of osteochondral defects,bone cysts, tumors, avascular necrosis and other related diseases orconditions.

The solid substrates, kits and methods of the invention may also be usedto enhance bone and/or cartilage formation in conditions where a boneand/or cartilage deficit is caused by factors other than bone remodelingdisorders. Such bone deficits include fractures, bone trauma, conditionsassociated with post-traumatic bone surgery, post-prosthetic jointsurgery, post plastic bone surgery, bone chemotherapy, post dentalsurgery and bone radiotherapy. Fractures include all types ofmicroscopic and macroscopic fractures. In one embodiment, some examplesof fractures includes avulsion fracture, comminuted fracture, transversefracture, oblique fracture, spiral fracture, segmental fracture,displaced fracture, impacted fracture, greenstick fracture, torusfracture, fatigue fracture, intra-articular fracture (epiphysealfracture), closed fracture (simple fracture), open fracture (compoundfracture) and occult fracture. In one embodiment, fractures meant to betreated using the methods of the present invention are non-unionfractures.

In one embodiment, methods of this invention are utilized for induced orenhanced repair of a cartilage and/or bone defect or disorder ordisease. In one embodiment, the cartilage defect results from a trauma,a tear, a sports injury, a full thickness articular cartilage defect, ajoint defect, or a repetitive stresses injury (e.g., osteochondralfracture, secondary damage due to cruciate ligament injury). In oneembodiment, the cartilage disorder comprises a disease of the cartilage.In one embodiment, methods of this invention induce or enhance cartilagerepair in osteoarthritis, rheumatoid arthritis, aseptic necrosis,osteochondritis dissecans, articular cartilage injuries, chondromalaciapatella, chondrosarcoma, chondrosarcoma-head and neck, costochondritis,enchondroma, hallux rigidus, hip labral tear, osteochondritis dissecans,torn meniscus, relapsing polychondritis, canine arthritis, fourthbranchial arch defect or cauliflower ear. In one embodiment, methods ofthis invention induce or enhance cartilage repair in degenerativecartilagenous disorders comprising disorders characterized, at least inpart, by degeneration or metabolic derangement of connective tissues ofthe body, including not only the joints or related structures, includingmuscles, bursae (synovial membrane), tendons, ligaments, and fibroustissue, but also the growth plate, meniscal system, and intervertebraldiscs.

In one embodiment, the solid substrates, kits and methods of theinvention may also be used to augment long bone fracture repair;generate bone in segmental defects; provide a bone graft substitute forfractures; facilitate tumor reconstruction or spine fusion; provide alocal treatment (by injection) for weak or osteoporotic bone, such as inosteoporosis of the hip, vertebrae, or wrist, or a combination thereof.In another embodiment, the solid substrates, kits and methods of theinvention may also be used in a method to accelerate the repair offractured long bones; treat of delayed union or non-unions of long bonefractures or pseudoarthrosis of spine fusions; induce new bone formationin avascular necrosis of the hip or knee, or a combination thereof.

In one embodiment, methods of this invention are evaluated by examiningthe site of cartilage and/or bone tissue repair, wherein assessment isby histology, histochemistry, palpation, biopsy, endoscopy, arthroscopy,or imaging techniques comprising X-ray photographs, computerized X-raydensitometry, computerized fluorescence densitometry, CT, MRI or anothermethod known in the art, or any combination thereof.

In one embodiment, a method of this invention comprises inducing andenhancing cartilage and/or bone repair wherein implanting a solidsubstrate of this invention within a site of cartilage and/or bonerepair influences and improves cartilage and/or bone repair.

In one embodiment, a method of this invention induces or enhancescartilage and/or bone repair, wherein the solid substrate attracts apopulation of cells to the solid substrate, thereby influencing orimproving cartilage and/or bone repair.

A clinician skilled in the art will recognize that methods of thisinvention, which entail implanting a coralline solid substrate within asite of cartilage and/or bone repair, may require preparation of a siteof cartilage and/or bone repair. These preparations may occur prior toimplantation of a coralline solid substrate or simultaneously withimplantation. For example, cartilage and/or bone tissue and/or othertissues proximal to a site of cartilage and/or bone repair may initiallybe drilled through to create a channel of dimensions appropriate for acoralline solid substrate used in the methods of this invention. Thenthe coralline solid substrate is implanted within the site so that aregion of the coralline solid substrate penetrates the drilled cartilageand/or bone tissues. Alternatively, the coralline solid substrate may beattached to a tool capable of penetrating through cartilage and/or boneor other tissues, or a combination thereof. In this case, as the toolpenetrates through the cartilage and/or bone tissue, the attachedcoralline solid substrate is simultaneously implanted.

In some embodiments, following implantation of the coralline solidsubstrate within a repair site, or several coralline solid substrateswithin the repair site, the coralline solid substrate is processed tooptimize incorporation and optimal cartilage and/or bone repair. In someembodiments, such processing may comprise cutting, sanding or otherwisesmoothing the surface of the coralline solid substrate or corallinesolid substrates, for optimal repair.

In some embodiments, the solid substrates as herein defined will becharacterized by a specific fluid uptake capacity value of at least 75%,which specific fluid uptake capacity value is determined by establishinga spontaneous fluid uptake value divided by a total fluid uptake value.

In some embodiments, the solid substrates as herein defined will becharacterized as having a contact angle value of less than 60 degrees,when in contact with a fluid.

In some embodiments, the solid substrates as herein defined will becharacterized by a structure such as that evident in FIGS. 8D-8F, whenassessed by scanning electron microscopy. In some embodiments, the solidsubstrates as herein defined will be characterized by a structure suchas that evident in FIGS. 9D-9F, when assessed by atomic forcemicroscopy.

In some embodiments, when a solid substrate is prepared by a processcomprising:

-   -   isolating a marine organism skeletal derivative-based solid        material;    -   establishing a specific fluid uptake capacity value of said        marine organism skeletal derivative-based solid material, which        specific fluid uptake capacity value is determined by        establishing a spontaneous fluid uptake value divided by a total        fluid uptake value; and    -   selecting a marine organism skeletal derivative-based solid        material characterized by a specific fluid uptake capacity value        of at least 75%, or in some embodiments, when a solid substrate        is prepared by a process comprising:    -   Isolating or preparing a organism skeletal derivative-based        solid material;    -   contacting said organism skeletal derivative-based solid        material with a fluid and establishing a contact angle for said        organism skeletal derivative; and    -   selecting a organism skeletal derivative-based solid material        characterized by a contact angle of less than 60 degrees;        or in some embodiments, such marine organism skeletal        derivative-based solid material will be characterized by a        substantial surface roughness (Ra) as measured by scanning        electron microscopy or atomic force microscopy.

In some embodiments, the present invention provides processes forconverting sub-optimized marine organism skeletal derivatives to marineorganism skeletal derivatives providing for cell or tissue growth orrestored function. In some embodiments, the present invention providesprocesses for optimizing marine organism skeletal derivatives providingfor enhanced cell or tissue growth or restored function. In someembodiments, according to this aspect, the initial isolation andprocessing of such marine organism skeletal derivatives so as to be in aform compatible with implantation within mammalian tissue, for example,in human or veterinary applications, may diminish the specific fluiduptake capacity value and the optimization processes of this inventionfacilitate improvement of such value.

According to this aspect, and in some embodiments, such isolation andprocessing of marine organism skeletal derivatives for use in accordancewith this invention includes exposure to a solution of sodiumhypochlorite and hydrogen peroxide. According to this aspect, it isstandard practice to treat coral/aragonite samples with sodiumhypochlorite as part of a first cleaning/processing protocol [see forexample, U.S. Pat. No. 5,433,751].

While various groups have proposed the use of coral-based materials intherapeutic applications in human subjects, there has been, to date, noindication that not all coral samples of a given species provide atherapeutic effect.

Surprisingly, it has now been found that there exists variability in thephysical characteristics of the coral, which impact its therapeuticapplication, and moreover, that certain standard processing stepsnegatively impact the therapeutic potential of coral-based materials.

Even more surprising is the finding herein that the therapeutic activitycan be restored and/or improved, by the application of certain processsteps, for example, the selective application of ethanol to the sampleas part of the processing steps of the coral-based materials of thisinvention.

In some embodiments, this invention provides a process for converting asuboptimal marine organism skeletal derivative-based solid substrate toan optimized marine organism skeletal derivative-based solid substratesfor promoting cell or tissue growth or restored function, said processcomprising:

-   -   a) establishing a specific fluid uptake capacity value for a        group of marine organism skeletal derivative-based solid        materials, which specific fluid uptake capacity value is        determined by establishing a spontaneous fluid uptake value        divided by a total fluid uptake value for each sample in said        group;    -   b) selecting a marine organism skeletal derivative-based solid        material characterized by a specific fluid uptake capacity        value;    -   c) contacting said marine organism skeletal derivative-based        solid material of (b) with an amphiphilic material, a polar        solvent, an a-polar solvent, a cationic material, an anionic        material, or a combination thereof;    -   d) determining a specific fluid uptake capacity as in (a) in        said marine organism skeletal derivative-based solid materials        obtained in (c); and    -   e) selecting marine organism skeletal derivative-based solid        materials obtained in (d) having a newly established increased        specific fluid uptake capacity value.

In some embodiments, the increased specific fluid uptake capacity valueis increased by at least 3%. In some embodiments, the increased specificfluid uptake capacity value is increased by at least 5%. In someembodiments, the increased specific fluid uptake capacity value isincreased by at least 4.5%. In some embodiments, the increased specificfluid uptake capacity value is increased by from at least 3%-15%.

According to this aspect, and in some embodiments, such a marineorganism skeletal derivative-based solid material is characterized by aspecific fluid uptake capacity value of between 75% and 95%.

In some embodiments, the increased specific fluid uptake capacity valueis increased by at least 10-15%. According to this aspect, and in someembodiments, such marine organism skeletal derivative-based solidmaterial is characterized by a specific fluid uptake capacity value ofbetween 45% and 70%.

In some embodiments, the increased specific fluid uptake capacity valueis increased by at least 20-35%. According to this aspect, and in someembodiments, such marine organism skeletal derivative-based solidmaterial is characterized by a specific fluid uptake capacity value ofbetween 1% and 40%.

In some embodiments, the amphiphilic material is a detergent orsurfactant. In some embodiments, the amphiphilic material is tween or anon-ionic copolymers composed of a central hydrophobic chain of forexample polyoxypropylene (poly(propylene oxide)) flanked by twohydrophilic chains of for example polyoxypropylene.

In some embodiments, the polar solvent is an alcohol, such as ethanol,methanol, acetone, isopropanol and others.

In some embodiments, the a-polar solvent is toluene, hexane, xylene andothers.

In some embodiments, the method further comprises the step of applying asecondary cleansing method to said marine organism skeletalderivative-based solid materials of (b), following or prior tocontacting the substrate with the materials listed in (c).

In some embodiments, the secondary cleansing method includes applyingpressure, heat, sonication, ethanol, organic solvent, salt buffer suchas phosphate buffer, steam treatment or a combination thereof.

In some embodiments, the secondary cleansing method includes treatmentwith an organic solvent. In some embodiments, the secondary cleansingmethod includes enzymatic treatment, such as, but not limited to, use ofpapain, trypsin or chondroitinase ABC. In some embodiments, thesecondary cleansing method includes sonication, heating, freeze drying,high pressure application, immersion under high pressure and the like.

In another embodiment, the invention provides a process for selection ofan optimized marine organism skeletal derivative-based solid substratefor promoting cell or tissue growth or restored function, said processcomprising:

-   -   Isolating or preparing a marine organism skeletal        derivative-based solid material;    -   establishing the presence of a substantially rough surface on        said marine organism skeletal derivative-based solid material,        which substantially rough surface is determined by scanning        electron microscopy or atomic force microscopy; and    -   selecting a marine organism skeletal derivative-based solid        material characterized by a determination of the presence of a        substantially rough surface on said marine organism skeletal        derivative-based solid material.

In some embodiments, according to this aspect, the process furthercomprises the step of contacting said marine organism skeletalderivative-based solid material with a fluid and applying negativepressure to said marine organism skeletal derivative-based solidmaterial to promote maximal uptake of said fluid within said marineorganism skeletal derivative-based solid material to arrive at saidtotal fluid uptake value.

In some embodiments, the invention provides a solid substrate producedby the process according to any aspect as herein described.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the solid substrates, kits,process and methods of the present invention without departing from thespirit or scope of the invention.

In one embodiment, the present invention provides combined preparations.In one embodiment, the term “a combined preparation” defines especiallya “kit of parts” in the sense that the combination partners as definedabove can be used independently or in different combinations i.e.,simultaneously, concurrently, separately or sequentially.

EXAMPLES Example 1 Physical Properties Variability in Coralline-BasedSolid Substrates of this Invention Materials and Methods

A diamond disk saw was used to remove an outer coral layer, and largesections from which representative smaller sections of desireddimensions were cut from the coral block.

Coral from the hydrocoral Porites lutea which has an average pore sizeof 100-150 μm was harvested from various regions within a coral. Thecoral was evaluated visually for its appearance, density, and porosity.Coral was then optionally immersed in 5% sodium hypochlorite for removalof external organic tissue. Briefly, coral was first exposed to a 5%sodium hypochlorite solution for 30 minutes, 3 exchanges at temperaturerange RT at 50° C., and sub-atmospheric pressure using vacuum pressureranging from 0.2-0.00001 Bar. The coral sections were then exposed to a10% solution of hydrogen peroxide for 15 minutes at a temperature rangeof from RT-50° C., and subatmospheric pressure using vacuum pressureranging from 0.2-0.00001 Bar. The cleaned sections were then washed indistilled water for 30 minutes, 3 exchanges at a temperature range offrom RT-50° C., and sub-atmospheric pressure using vacuum pressureranging from 0.3-0.00001 Bar.

The coral was optionally sterilized by exposure to gamma radiation at astrength of at least 22.5 kGy and can then be stored aseptically, inpackaging material, and in particular, the smaller samples wereirradiated, whereas larger blocks assessed were not irradiated.

Each section was then place in a plastic petri dish and 2 ml of fluidwas applied to each dish. Observations regarding absorption of the fluidwere recorded. Fluids used included animal blood, plasma, water andvarious colored solutions.

Results

To determine whether sample removal from various regions provides formaterials, which vary in their physical characteristics and whether suchvariability provides for alternative qualities to the same, blood andother fluids listed were applied to the coral samples and absorption ofthe fluid within the coral samples was assessed.

FIGS. 1A-1F depict the results of a representative absorption studyconducted as described. Coral samples were isolated from differentregions of a coral block, and assessed for their pattern and intensityof absorption of blood applied thereto. Surprisingly, there appears tobe no uniformity in terms of the absorption profile, and the same is notan “all or none” phenomenon.

FIG. 1A for example, shows reasonably substantial absorption throughoutthe structure, whereas FIG. 1C shows poor to no absorption throughout,and FIG. 1B provides an interim phenomenon within the structure in thatsome regions substantially absorb the fluid and some regions absorbminimal to no fluid. FIGS. 1D-1F show cross-sectional slices throughcoral pieces from which coral plugs were cut and prepared, providingdifferent patterns of absorption within the macrostructures, as well.

Other fluids were assessed in terms of their absorption within parallelsamples comparable to the sample in FIG. 1C. To serve as a stain, a saltsolution, and protein solution, carbohydrate solutions, ionic solutionswere prepared and applied, and the results substantially mirrored thatof the applied blood in that poor to no absorption occurred in thesample of FIG. 1C. Plain water applied thereto provided substantiallythe same, resulting in poor to no absorption within the coral sample ofFIG. 1C.

FIGS. 1D-1F provide images of larger blocks of coral from which thesamples of FIGS. 1A-1C were taken, respectively. As can be seen in FIG.1D, the region from which the sample of 1A was taken shows good uptakeof the fluid applied, in this case, blood, whereas the region from whichthe sample was taken in FIG. 1C (i.e. the block of FIG. 1F) showsminimal uptake, and the region from which the sample of FIG. 1B wastaken, shown in FIG. 1E shows an intermediate uptake, in that someregions show good uptake, whereas other regions show minimal uptake.

As demonstrated herein, the size of the sample assessed is not limiting,and indeed samples of various sizes and thicknesses may be thusassessed. Furthermore, differences in surface tension are evident(compare FIG. 1C to FIG. 1A).

Example 2 Establishing a Screening Protocol for Coralline-Based SolidSubstrates

Based on the findings in Example 1, a screening protocol may beestablished to select for an optimized coral-based solid substrate forpromoting cell or tissue growth or restored function. FIG. 2 provides aflow diagram of an envisioned screening protocol process. Coral samplesare identified, isolated and machined to a desired size and shape, orassessed in blocks. The samples are then cleaned and optionallysterilized, then dried. The coral sample being assessed may be driedunder vacuum and/or heated toward this end.

A dry weight for each sample may then be recorded.

Fluid as described herein is added to each assay container in anapproximately 1:1 ratio or slightly more, i.e. equal to or slightly morethan the size of the sample in mm as compared to the volume of fluid inml is added to the container.

The sample may then be weighed and a spontaneous fluid uptake value isdetermined.

Samples may optionally be dried, prior to further manipulation of thesample.

A significantly increased amount of fluid is brought into contact withthe sample and a vacuum is applied for a period of time to ensuremaximal uptake of the applied fluid into the coral sample.

A total fluid uptake capacity is assessed and the specific fluid uptakecapacity value is determined by dividing the spontaneous fluid uptakevalue by the total fluid uptake capacity.

If the value exceeds the cutoff value of 75%, then the sample will benoted for its suitability as a solid substrate for promoting cell ortissue growth or restored function. When the sample is utilized in vivoin subsequent applications, in some aspects, the sample is firstcontacted with autologous biological fluids or materials from the hostprior to implantation into the same. If the value is less than thestated cutoff value, then the sample is not used as a solid substratefor promoting cell or tissue growth or restored function.

Example 3 Improved Solid Substrate Incorporation as a Function ofCertain Physical Properties in Coralline-Based Solid Substrates of thisInvention

In order to assess the consequence of the phenotypic variability inblood absorption in the plugs of Example 1, coral plugs were preparedusing a standard production method including three hypochlorite washes,hydrogen peroxide treatment and multiple double distilled water washes.Their spontaneous fluid uptake and total fluid uptake values weredetermined as described in Example 2, with water being the sample fluidassessed in this case. Sample implants exhibiting a spontaneous fluiduptake value of more than 75% were also checked for their spontaneousblood uptake ability.

Implants were graded as red, white, and intermediate, with intermediatereferring to regions that are red and regions that remain white.Implants were placed in each goat knee so that each goat receivedimplants characterized by a spontaneous fluid uptake value of more than75% and implants which were characterized by a spontaneous fluid uptakevalue of less than 50%. The animals were followed for 4 weeks and thensacrificed.

Early cartilage formation was assessed macroscopically andhistologically. Osteointegration and early bone formation or resorptionwas assessed using X-ray, micro-CT and histology

FIGS. 3A-3F demonstrate the correlation between biologic fluid uptakebefore implantation and site healing over time. Implants characterizedby significant water and blood uptake, or minimal uptake thereof withinthe implant, were implanted within a defect site and evaluatedmacroscopically, at 4 weeks post-implantation. Tissue consistent withhyaline cartilage appearance substantially covered the implant, insamples with significant fluid uptake, whereas samples which werecharacterized by minimal/diminished fluid uptake presented with a morefibrous capsule covering over the implant implantation (FIG. 3A versus3D, respectively). X-ray and micro-CT analysis of the respectiveimplants characterized by minimal/diminished fluid uptake [FIGS. 3B and3C] versus those characterized by significant fluid uptake [FIGS. 3E and3F] demonstrated that implants characterized by significant fluid uptakeappear to be properly integrated within the implantation site with nosignificant adverse reaction. Implants characterized byminimal/diminished fluid uptake appear to induce bone resorption, lysisand loosening of the implant.

Example 4 Prescreening Coralline-Based Solid Substrates for Implantation

For applications in promoting cell or tissue growth or restoredfunction, solid substrates are assessed for their ability to absorbfluid, such as, for example, water. Substrates that provide a specificfluid uptake capacity value of at least 75% are then implanted in thedesired tissue site. For example, and representing one embodiment, sucha solid substrate may be implanted within cartilage and neighboring bonefor applications in repairing or regeneration in osteochondral defectsor disease.

Solid substrates may be prepared according to any embodiment asdescribed herein, as will be appreciated by the skilled artisan.

The substrates are envisioned for use in veterinary applications, aswell as in the treatment of human subjects. At appropriate intervals,standard methodology is employed to assess good incorporation of thesubstrates and healing of the affected tissue, for example, X-ray, CT orMRI imaging may be performed to verify the position of the implants.

Implantation may be at any suitable location, for example, for kneejoint repair, implantation may be within the Medial Femoral Condyle(MFC), Lateral Femoral Condyle (LFC), Patela, Trochlear Groove (TG) andthe Tibia.

In applications relating to promoting cell or tissue growth or restoredfunction, it is noted that solid substrates characterized by a specificfluid uptake capacity value of at least 75% will significantlyoutperform solid substrates characterized by a specific fluid uptakecapacity value of less than 40%, in terms of their ability to promotecell or tissue growth or restored function at the site, promote healingat the implantation site, promote returned tissue function, or acombination thereof.

Example 5 Physical Properties Variability in Coralline-Based SolidSubstrates of this Invention

Natural surfaces are heterogenic due to their variable materialcomposition, surface roughness and porosity and thus demonstratevariable water repellence/adhesion characteristics. Contact anglemeasurements can characterize the wetting of rough surfaces, taking thetopography and the chemical structure of the surface into account.

Contact angle measurement was with goniometry. The contact angle is anequilibrium contact angle measured macroscopically on a solid surface.The same is to be distinguished from Young contact angles, measured onatomically smooth, chemically homogeneous surfaces.

The regions were classified into three classes and their relativesurface areas were approximated as a percentage out of the total surfacearea:

Regions characterized by contact angles of between 0 and 60 deg, appearas white regions in the figures provided. Regions characterized by acontact angle of between 60 and 90 deg and regions characterized by acontact angle of 90 deg and higher are shaded in the figures provided.

Water drops of 1 ul-10 ul volume were deposited on cleaned and driedcoral samples with a precise micro-dosing syringe. The contact angleswere measured with a Rame-Hart goniometer (Model 500) with an accuracyof 0.1 deg (Bormashenko, 2012). Measurements were assessed for bothsides of the drop and averaged. The test medium employed was physiologicsaline.

Three 3×3 mm coral samples termed, R43, R34, and R44 were assessed.Prior to evaluation of contact angles, the specific fluid uptakecapacity value was assessed for samples from each block, and the resultsare presented in Table 1.

Specimen specific fluid uptake Coral (Name) capacity value 43 1 0.62 20.46 3 0.60 4 0.31 por1 0.17 por2 0.17 por3 0.37 44 1 0.32 2 0.82 3 0.8834 1 0.70 2 0.39 3 0.33

FIGS. 4A-4D provide photographs of coral sample R43 specimens evaluatedfor their contact angles. FIGS. 4A and 4B show regions cut from thelarger block, which were assessed for their contact anglecharacterization. The majority of regions of the block assessed in FIGS.4A and 4B provided for a contact angle primarily of less than 60degrees. Certain areas in FIGS. 4C and 4D provided for a contact angleof between 60 and 90 degrees (FIG. 4C) and over 90 degrees (FIG. 4D).

FIGS. 5A-5C provide provides photographs of coral sample R34 specimensevaluated for their contact angles. FIG. 5A shows regions cut from thelarger block, which were assessed for their contact anglecharacterization. The majority of regions of the block assessed in FIGS.5B and 5C provided for a contact angle primarily of less than 60degrees. Certain areas in FIGS. 5B and 4C provided for a contact angleof between 60 and 90 degrees and over 90 degrees. FIGS. 6A and 6Bsimilarly provide photographs of coral sample R44 specimens evaluatedfor their contact angles. FIG. 6A shows regions cut from the largerblock, which were assessed for their contact angle characterization. Themajority of regions of the block assessed in FIG. 6B provided for acontact angle primarily of less than 60 degrees. Certain areas in FIG.6B provided for a contact angle of between 60 and 90 degrees and over 90degrees.

The contact angle measurements parallel the specific fluid uptakecapacity values obtained for respective coral samples. Accordingly, theimproved solid substrates for promoting cell or tissue growth orrestored function of this function may be characterized by either adetermination of a contact angle, or a specific fluid uptake capacityvalue.

Furthermore, environmental scanning electron microscopy (ESEM) studiesconfirmed the results of the contact angle studies presented herein.

Table 2 presents the specific fluid uptake capacity values for the coralsamples evaluated by ESEM.

Specimen specific fluid uptake Coral (Name) capacity value R27 7 0.87R30 40 0.04 R43 1 0.62

FIGS. 7A-7E present the results of ESEM analysis conducted on thesamples described in Table 2. Samples assessed from the R27-7 blockprovided for a zero drop angle value, and no drop formation seen (FIG.7A). FIG. 7B-7C present the results for sample R30-40. FIG. 7B was takenfollowing application of fluid, and it was noted that the sample failedto “wet” when water was applied. FIG. 7C shows that followingre-desiccation, water droplets were evident on the surface, consistentwith a phenotype of poor surface wetting.

FIGS. 7D-7E present the results for sample R43-1, with resultsconsistent with those seen for sample R30-40.

Taken together, these results are corroborative of the contact angledata, as well specific fluid uptake capacity values obtained forrespective coral samples. A sample having a specific fluid uptakecapacity values obtained for respective coral samples of more than 75%exhibited no drop formation on the surface, consistent with a “goodwetting” phenotype (FIG. 7A), whereas samples with a lower specificfluid uptake capacity value exhibited droplet formation duringdessication.

Example 6 Physical Characteristics of Improved Coralline-Based SolidSubstrates of this Invention

Coral samples were processed as described in Example 1, hereinabove.Samples were then subjected to environmental scanning electronmicroscopy and atomic force microscopy, according to standard methods.

FIGS. 8A-8C demonstrate the surface structure of a solid substrate witha minimal specific fluid uptake capacity value as compared to that ofsubstrates with a substantial specific fluid uptake capacity value(compare FIGS. 8A-8C and 8D-8F). Substrates with a lower specific fluiduptake capacity value exhibited a smoother outer surface structure, ascompared to those with a higher specific fluid uptake capacity value,while the crystalline structure of the latter sample was easily seen.

Furthermore, atomic force microscopy demonstrated that substrates with alower specific fluid uptake capacity value were characterized by asmoother outer surface (FIGS. 9A-9C). In marked contrast, substratescharacterized by a higher specific fluid uptake capacity value,exhibited a rougher surface (FIGS. 9D-9F).

Taken together, these results are corroborative of the fact that surfacestructure characterization correlates with specific fluid uptakecapacity values obtained for respective coral samples. A sample having aspecific fluid uptake capacity values obtained for respective coralsamples of more than 75% exhibited a rougher surface, whereas sampleswith a lower specific fluid uptake capacity value exhibited a smoothersurface.

Example 7 Development of a Process for Converting a Suboptimal Group ofMarine Organism Skeletal Derivative-Based Solid Substrates to OptimizedMarine Organism Skeletal Derivative-Based Solid Substrates Materials andMethods

Coral samples of the hydrocoral Porites lutea were isolated as describedin Example 1 and plugs were prepared.

Plugs were then weighed, establishing a dry weight per sample. Plugswere exposed to 2 ml of double distilled water for 5 minutes, thenweighed, providing a spontaneous uptake value. Plugs were then exposedto an excess of double distilled water for 30 minutes under vacuum andthen weighed, to determine the total fluid uptake value for each plug.The specific fluid uptake capacity value was then determined by dividingthe spontaneous fluid uptake values by the total fluid uptake valuesobtained.

Each section was optionally placed in a plastic petri dish and 2 ml offluid was applied to each dish, such as blood, and the respectivephenotype of fluid uptake was preserved in samples which yielded aspecific particular fluid uptake capacity value.

Samples shown to yield poor uptake and having a specific fluid uptakecapacity value of less than 40%, samples shown to yield an intermediateoverall specific fluid uptake capacity value of between 41% and 74% andsamples shown to yield a specific fluid uptake capacity value of between75% and 99% were then contacted with test materials. In some aspects,the test conditions included the application of 5 ml of 0.5% Tween 80.

In some aspects, the test conditions included sonication for 15 minutes;application of absolute ethanol; application of 5 ml pluronic with orwithout prior tween 80 application. In some aspects, the test conditionsincluded the application of 5 ml of Methylene blue in 0.03% acetic acidor 3M 0.05% hyaluronic solution in DDW.

Following such treatments, the samples were then contacted again with afluid as described above, such as water, and a specific fluid uptakecapacity value was assessed again for each sample. Uptake of a fluidwithin the plug, such as uptake of blood was verified macroscopically,as well.

Samples providing a specific fluid uptake capacity value of more than50% were similarly evaluated and compared to those with a value of lessthan 50%.

Results

Example 2 demonstrated that solid substrates characterized by a specificfluid uptake capacity value of at least 75% provide for cell or tissuegrowth or restored function and in a given isolated block of a marineorganism skeletal derivative, there is variability in terms of thenumber of plugs that can be isolated therefrom, exhibiting the desiredspecific fluid uptake capacity value. It was therefore of interest todetermine whether samples characterized by a specific fluid uptakecapacity value of less than 40%, or of between 41% and 74%, or ofbetween 75% and 99% could be increased, improving each substratesability to provide for cell or tissue growth or restored function.

In order to determine if it were possible to isolate samples of suchskeletal derivatives characterized by the indicated ranges for eachspecific fluid uptake capacity value, respectively, and to treat thesample to thereby improve the specific fluid uptake capacity value to begreater than that previously obtained, a number of manipulations of thesample were undertaken.

Tween 80 is a non-ionic surfactant and emulsifier derived frompolyethoxylated sorbitan and oleic acid. Tween 80 was therefore assessedfor its ability to alter the specific fluid uptake capacity value of thecoral samples tested. FIG. 10 provides the results of a number ofsamples assessed. In essentially every sample assessed, tween treatmentincreased the specific fluid uptake capacity (SFUC) value. Some samplesexhibiting lower SFUC values were significantly enhanced. Sample 31-10,35-5 and others, exhibited an initial SFUC value of less than about 30%and showed a marked increase in SFUC following tween 80 treatment, tovalues as high as over 60% and even over 80%. When blood was applied tothe samples, rapid uptake occurred, in marked contrast to prior pooruptake (FIG. 10). Since treatment of the plugs with an amphiphiliccompound provided for increased SFUC in all samples evaluated, it was ofinterest to determine whether other manipulations would improve thisphenomenon.

Toward this end, samples to which Tween 80 had been applied were thensubjected to sonication, as well. FIG. 11 demonstrates the results ofsuch process. As can be seen in the figures, in most samples, the SFUCobtained with tween 80 treatment and sonication improved the subsequentSFUC value. Sample 37-30, 37-14 and others, exhibited an SFUC increaseto more than double the initial value In a number of samples, tween 80and sonication treatment resulted in SFUC values of more than 85%.

In order to determine whether other amphiphilic compounds would providethe same results as that seen with tween in terms of increasing an SFUCvalue, pluronic (non-ionic tri-block copolymers composed of a centralhydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked bytwo hydrophilic chains of polyoxypropylene) was applied to the samples,instead of tween, in accordance with the methods described hereinabove.As can be seen in FIG. 12, treatment with pluronic increased SFUC inessentially every sample tested (compare light grey bars with darkergrey bars in FIG. 12). In most cases, applying both pluronic and tween80 to the samples further increased the SFUC value obtained.

In order to determine whether polar solvents would provide the sameresults as that seen with tween in terms of increasing an SFUC value,absolute ethanol was applied to the samples. FIG. 13 demonstrates thatas was seen with tween and pluronic, when ethanol was applied a largenumber of the samples evaluated showed an increase in SFUC, although inthis case, the increase was less substantial when samples with aninitial SFUC of more than 70% were evaluated.

In order to determine whether cationic compounds would provide the sameresults as that seen with tween in terms of increasing an SFUC value,samples were exposed to methylene blue, similarly to that described forTween 80 in the methods section hereinabove. As can be seen in FIG. 14,indeed application of methylene blue to samples paralleled thephenomenon observed with ethanol somewhat, in that the greater increaseswere found with samples having an SFUC value of less than 30%.

In order to determine whether anionic compounds would provide the sameresults as that seen with tween in terms of increasing an SFUC value,samples were exposed to hyaluronic acid, similarly to that described forTween 80 in the methods section hereinabove. As can be seen in FIG. 15,indeed application of hyaluronic acid to samples increased SFUC valuesin most samples evaluated, however, such increases were more modest thanthat assessed for Tween 80.

Similarly, as can be seen in FIG. 16, when chondroitin sulphate wasused, an increase in SFUC was evident in each sample.

To further the results reported hereinabove, a series of implantsexhibiting a specific fluid uptake capacity value of at least 75% (n=42)from 3 different coral slabs (R-76, R-77, R-78) were subjected to apurification process, as follows: samples were immersed in a 5% NaOCl(W/W) solution at a ratio of 1:20 (plug volume:NaOCl) for 30 minutesunder an applied negative pressure of at least 0.03 bar, after which theliquid was decanted and samples were exposed to applied negativepressure of at least 0.03 bar for 30 additional minutes. Samples werethen immersed and immersed in 10% (W/W) H₂O₂ solution at a ratio of 1:20(plug volume:H₂O₂) under an applied negative pressure of at least 0.03bar for 15 minutes, after which the liquid was decanted and samples wereexposed to applied negative pressure of at least 0.03 bar for 30additional minutes. Samples were washed repeatedly in water, followed bybeing immersed in sterile water at a ratio of 1:20 (plug volume:H2O) andthen exposed to applied negative pressure of at least 0.03 bar for 30additional minutes. The wash step was repeated at least three times.Samples were then dried under a vacuum pressure of 0.03 bar for at least4 to 6 hours. The specific fluid uptake capacity value was thenascertained for representative samples.

Samples were then immersed in absolute ethanol at a ratio of 1:10 (plugvolume:ethanol) under vacuum conditions of at least 0.03 bar for 30minutes, followed by decanting of the solution, and washes in sterilewater as described in the previous paragraph. The specific fluid uptakecapacity value (SWC) was then ascertained for representative samples andthe results are provided in FIG. 17, showing the average SWC values ofthe indicated samples. As is evident from the figure, the SWC valueimproved in a statistically significant manner when the basic cleaningprotocol was followed by an extraction step with a polar solvent, inthis case, ethanol.

FIGS. 18-19 show the average SWC values obtained for numerous samples,before and after an ethanol extraction step applied following the basicisolation/processing step. As is clear from the myriad of samplesassessed, unexpectedly, the specific fluid uptake capacity value isincreased following the basic isolation/processing step, when anextraction step is executed using a polar solvent, such as ethanol.

Example 8 Automated Process and Apparatus for Isolating and PreparingOptimized Marine Organism Skeletal Derivative-Based Solid Substrates

Coral samples of the hydrocoral Porites lutea are isolated as describedin Example 1 and implants are prepared. The implants 20-10 are placed inthe holding cassette 20-20, and the cover 20-70 is placed on the device.Negative pressure is applied via the vacuum 20-30 for a prescribedperiod of time, until the implants are fully dry and then theapplication is halted. The apparatus then individually weighs eachimplant to establish a dry weight. An automated cycle is initiated,which facilitates filling and a desired fluid level is maintained duringthe process. The cassettes 20-20 are individually raised and loweredinto a first fluid level, via the cassette manipulator 20-40 within thefluid to enable spontaneous fluid uptake, and the implant manipulator20-60 individually orients/moves the individual implants for weightdetermination, to determine the spontaneous fluid uptake value followedby optionally a pass of each implant past a drying/blotting station. Theindividual implants are all weighed and returned to their place withinthe cassettes. The cassettes 20-20 are then again individually raisedand lowered into a second, significantly higher fluid level, via thecassette manipulator 20-40 within the excess fluid to facilitate fullimmersion of the implants, and negative pressure is applied again viathe vacuum 20-30 for a defined period of time, ensuring maximum fluiduptake within each implant. The implant manipulator 20-60 againindividually orients/moves the individual implants for a second weightdetermination, which provides the total fluid uptake value. The dataprocessing unit of the apparatus determines and provides an output ofthe specific fluid uptake value, optionally specifically identifyingwhich samples are to be selected based on indicated criteria.

It will be understood that the dimensions of the cassette will beconstructed to accommodate implants of varying size. The apparatus canbe built to scale, as well, to accommodate a larger or smaller number ofcassettes, and the materials will be appropriate for the various fluidsbeing assessed for their uptake within the stated implants. Sensors andappropriate relays are incorporated to, for example, provide a warningsystem in case of malfunction and the apparatus may further comprise adata processing unit, to calculate the specific fluid uptake capacityvalue from the determined spontaneous and total fluid uptake valuesobtained. Statistical analysis may also be included as part of the dataprocessing package provided optionally with the claimed apparatuses ofthis invention.

Example 9 Improved Cell Adhesion and Viability with Optimized MarineOrganism Skeletal Derivative-Based Solid Substrates Materials andMethods

Human Embryonic Palatal Mesenchyme (HEPM) cells were grown inappropriate growth medium.

Implants of optimized (having a specific fluid uptake capacity value ofmore than 75%) and nonoptimized coral (having a specific fluid uptakecapacity value of less than 60%) of each coral were seeded with HEPMcells at a density of 1.65×10⁴ and 0.8×10⁴ cells/10 μl, respectively, byincubating the cells with the scaffold for 15 min at 37° C., followed bythe addition of more medium and further incubation for up to 7 days.Growth medium was replaced every 2 days.

Scaffolds containing cells were fixed in 4% formaldehyde, washed anddried via ethanol gradient solutions, and HMDS gradient solutions, thenassessed by SEM.

Cell attachment and morphology was observed by SEM at day 1, 3, and 7post seeding, following fixation.

Cell viability was assessed with an alamarBlue® metabolic assay,following the manufacturer's protocol. Samples were taken at day 1 and 7post seeding. Fluorescence was evaluated at 544 nm and 590 nm(excitation and emission, respectively) using Fluoreskan ascent, amicroplate fluorescence reader (Labotal), data in triplicates.

F fresh 200 l alamarBlue® containing medium (1:10 respectively) wasadded to each scaffold and incubate for 18 more hours (total of 24hours).

Results

Cell attachment was found as early as 1 day post seeding in all samplesevaluated, where optimized (opt), i.e. having a specific fluid uptakecapacity value of more than 75%, and nonoptimized (nonopt), i.e. havinga specific fluid uptake capacity value of less than 60%, as evaluated bySEM analysis.

Non-optimized samples, having a specific fluid uptake capacity value of26% showed that the cells adhered to the coral, with visible cellextensions (pods) proximally located the coral surface (FIG. 21A).Although cellular extensions make contact with coral substrate, themajority of the cell body does not seem to readily make contact. FIG.21B presents a higher magnification of the “boxed” area in FIG. 21A,showing contact of the terminal cellular extensions with the coral, butthe cell is not flattened or in full contact over its entire cell bodywith the coral.

In marked contrast, optimized samples, having a specific fluid uptakecapacity value of 95% showed that the cells adhered well to the coral,including full cell body spread over the coral (FIG. 21C).

HEPM cells seeded on non-opt and opt coral samples were assessed byalamarBlue® assay, which quantitates the proliferation of various humanand animal cell lines. At 1 day post seeding, values are normalized tothose obtained for cultures of cells seeded on a polystyrene well,alone. 100% is determined as the cell viability value on the polystyrenewell at day 1 (FIG. 22A). FIG. 22B represents HEPM cell viability on thevarious corals 7 days post seeding. In each coral, the values of the7-day were divided by the values of the 1-day, so that the fold increasein cell viability is shown. As is readily appreciated, optimized samplesyielded a significantly increased cell viability value compared tonon-optimized samples. The HEPM cell viability increased with an averageof 42 times higher in day 7 compared to day 1, whereas the increases inthe low SWC coral R29 and the control group were only 13 and 7 times,respectively.

Taken together, the cell adhesion and cell viability assays demonstratethat samples considered to be optimized for fluid uptake promote greaterfull cell adherence and viability over time.

Example 10 Crystalline Form Association with the Isolation andProcessing of Implants to Yield Optimized Marine Organism SkeletalDerivative-Based Solid Substrates Materials and Methods

Coral samples were isolated and prepared in accordance with the methodsdescribed for Example 1. Samples were stained according to the protocolas described in [Chemical staining methods used in the identification ofcarbonate minerals, Tamer AYAN, Mineral Research and ExplorationInstitute of Turkey http://www.mta.gov.tr/v2.0/eng/dergi_pdf/65/11.pdf].Briefly, Feigl solution is prepared according to standard methods. Thecoral samples are then stained with Feigl solution, and samplespreviously assessed for their specific fluid uptake capacity value arestained therewith. In this case, 2 samples as assessed in Example 9, aSample R91, having a specific fluid uptake capacity value of 95%, and aSample R48, having a specific fluid uptake capacity value of 42% wereassessed.

Results

Corals are composed mainly of calcium carbonate (˜98%). The calciumcarbonate can be in different crystalline forms such as aragonite andcalcite. Coral polyps may secrete an aragonite skeleton beneath theirbasal ectoderm forming a complex exoskeleton, which represents achronological layered archive. In some regions, the aragonite structuredissolves and calcite or micrite is formed, usually at a later stage, bya process known as Diagenesis.

It has been shown that more calcite precipitates are found in ancientcorals (Miocene epoch) compared to modern corals. FIG. 23A shows Miocenecoral stained with Feigl solution, where the black region represents thearagonite crystalline structure. FIG. 23B shows a coral stained by Feiglstaining, when no aragonite was present in the sample since no blackcolor is observed [Both figures are taken from Diagenesis of growthbands in fossil scleractinian corals: identification and modes ofpreservation, Reuter et al. Facies (2005), 51: 146-159, which referenceis fully incorporated herein in its entirety].

Feigl's solution stains aragonite black. When coral samples are viewedmacroscopically, in the absence of aragonite in a given sample, thesample will appear white or gray in color.

It was therefore of interest to determine whether the crystal structurecorrelated with the specific fluid uptake capacity value and whether theoptimization processes herein were impacted in terms of the Feiglstaining protocol. Toward this end, coral samples R91 and R48 wereassessed for their specific fluid uptake capacity value, including afirst macroscopic evaluation of uptake of goat blood, and subjected tofurther processing, including an ethanol extraction step as described inExample 7.

FIGS. 23C-23F present the results of analysis of essentially identicalsamples taken from R91 and R48, both prior to and following an ethanolfurther purification step. Whereas the gross pattern of Feigl stainingapproximated that of the uptake of blood in each respective sample(compare FIG. 23C versus FIG. 23D and FIG. 23E versus FIG. 23F), thestaining patterns are not identical. Moreover, as is more readilyobserved when comparing samples of R48 before and after ethanoltreatment, increased blood uptake and Feigl positive staining wasobserved, indicating greater optimization of the samples, when subjectedto a described further processing step.

Taken together, it appears that samples enriched for aragonite versusnon-aragonite are more readily associated with a higher specific fluiduptake capacity value, spontaneous blood absorption and black stainusing Feigl solution, which can be further improved by the specificfurther processing steps, as herein described.

It will be understood by those skilled in the art that various changesin form and details may be made therein without departing from thespirit and scope of the invention as set forth in the appended claims.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed in the scope of the claims.

In some embodiments, the term “comprise” or grammatical forms thereof,refers to the inclusion of the indicated components of this invention,as well as inclusion of other active agents, and pharmaceuticallyacceptable carriers, excipients, emollients, stabilizers, etc., as areknown in the pharmaceutical industry.

In one embodiment of this invention, “about” refers to a quality whereinthe means to satisfy a specific need is met, e.g., the size may belargely but not wholly that which is specified but it meets the specificneed of cartilage repair at a site of cartilage repair. In oneembodiment, “about” refers to being closely or approximate to, but notexactly. A small margin of error is present. This margin of error wouldnot exceed plus or minus the same integer value. For instance, about 0.1micrometers would mean no lower than 0 but no higher than 0.2. In someembodiments, the term “about” with regard to a reference valueencompasses a deviation from the amount by no more than 5%, no more than10% or no more than 20% either above or below the indicated value. Inone embodiment, the term “about” refers to a variance of from 1-10%, orin another embodiment, 5-15%, or in another embodiment, up to 10%, or inanother embodiment, up to 25% variance from the indicated values, exceptwhere context indicates that the variance should not result in a valueexceeding 100%.

In the claims articles such as “a”, “an” and “the” mean one or more thanone unless indicated to the contrary or otherwise evident from thecontext. Claims or descriptions that include “or” or “and/or” betweenmembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention also includes embodiments in which more than one, or all ofthe group members are present in, employed in, or otherwise relevant toa given product or process. Furthermore, it is to be understood that theinvention provides, in various embodiments, all variations,combinations, and permutations in which one or more limitations,elements, clauses, descriptive terms, etc., from one or more of thelisted claims is introduced into another claim dependent on the samebase claim unless otherwise indicated or unless it would be evident toone of ordinary skill in the art that a contradiction or inconsistencywould arise. Where elements are presented as lists, e.g. in Markushgroup format or the like, it is to be understood that each subgroup ofthe elements is also disclosed, and any element(s) can be removed fromthe group. It should be understood that, in general, where theinvention, or aspects of the invention, is/are referred to as comprisingparticular elements, features, etc., certain embodiments of theinvention or aspects of the invention consist, or consist essentiallyof, such elements, features, etc. For purposes of simplicity thoseembodiments have not in every case been specifically set forth in haecverba herein. Certain claims are presented in dependent form for thesake of convenience, but Applicant reserves the right to rewrite anydependent claim in independent format to include the elements orlimitations of the independent claim and any other claim(s) on whichsuch claim depends, and such rewritten claim is to be consideredequivalent in all respects to the dependent claim in whatever form it isin (either amended or unamended) prior to being rewritten in independentformat.

What is claimed is: 1.-118. (canceled)
 119. A process of promotingcartilage and/or bone repair or repair of an osteochondral defect, theprocess comprising implanting, in a subject, a solid substrate within asite in need of cartilage and/or bone repair, or in need of repair of anosteochondral defect, wherein the solid substrate is a marine organismskeletal derivative-based solid material, and wherein the specific fluidupdate capacity of the solid substrate is at least 75%.
 120. The processof claim 119, wherein the marine organism skeletal derivative-basedsolid material is a coral or a coral-based derivative.
 121. The processof claim 119, wherein the marine organism skeletal derivative-basedsolid material contains ground particles derived from coral, suspendedin a biocompatible matrix or further comprises a bone filler or bonesubstitute material.
 122. The process of claim 121, wherein thebiocompatible matrix is a hydrogel.
 123. The process of claim 119,wherein the marine organism skeletal derivative-based solid material isa bone filler or bone substitute material.
 124. The process of claim119, wherein the marine organism skeletal derivative-based solidmaterial comprises a biocompatible polymer.
 125. The process of claim124, wherein the biocompatible polymer is incorporated within voids orpores in the solid substrate or wherein the biocompatible polymer isattached to an outer surface of the solid substrate.
 126. The process ofclaim 124, wherein the biocompatible polymer comprises a natural polymercomprising a glycosaminoglycan, collagen, fibrin, elastin, silk,chitosan, alginate, and any combination thereof.
 127. The process ofclaim 126, wherein the biocompatible polymer comprises aglycosaminoglycan and the glycosaminoglycan is hyaluronic acid, sodiumhyaluronate, cross linked hyaluronic acid, or a combination thereof.128. The process of claim 119, wherein the marine organism skeletalderivative-based solid material further comprises a cytokine, a growthfactor, a therapeutic compound, an osteoinductive agent, a bioactiveglass, a bone filler, a bone cement, a drug, or any combination thereof,wherein the therapeutic compound or drug comprises an anti-inflammatorycompound, an anti-infective compound, a pro-angiogenic factor or acombination thereof.
 129. The process of claim 119, wherein the marineorganism skeletal derivative-based solid material approximates the formof a cylinder, cone, tac, pin, screw, rectangular bar, plate, disc,pyramid, granule, powder, coral sand, condyle, rib, pelvis, vertebra,bone, cartilaginous tissue, ball or cube.
 130. The process of claim 119,wherein the marine organism skeletal derivative-based solid materialapproximates a shape that accommodates a site of desired tissue growthor repair.
 131. The process of claim 119, wherein the marine organismskeletal derivative-based solid material comprises a hollow or hollowsalong a Cartesian coordinate axis of the coralline-based solid material.132. The process of claim 119, wherein the process further comprises thestep of contacting the marine organism skeletal derivative-based solidmaterial with cells or tissue.
 133. The process of claim 132, whereinthe process comprises contacting the marine organism skeletalderivative-based solid material with cells and the cells comprise stemor progenitor cells or a combination thereof.
 134. The process of claim119, wherein the subject is a human subject.
 135. The process of claim119, wherein the subject is a veterinary subject.
 136. The process ofclaim 119, wherein the subject is afflicted with a defect or disorder ordisease of the cartilage or bone or a combination thereof.
 137. Theprocess of claim 136, wherein the cartilage defect or disorder ordisease comprises a full or partial thickness articular cartilagedefect; osteochondral defect; osteoarthritis, a joint defect or a defectresulting from trauma, sports, or repetitive stress.
 138. The process ofclaim 136, wherein the defect or disorder or disease of the bonecomprises a fracture, bone defect, bone edema, osteoporosis, bone tumor,bone cyst, or a defect resulting from trauma, sports, or repetitivestress.